Nitrogen-containing 6-membered cyclic compound

ABSTRACT

A novel compound represented by the following general formula (1), or a salt thereof, which has a superior EP 4  receptor agonist activity, and a medicament containing the compound or a salt thereof as an active ingredient, which can be used for promotion of osteogenesis, therapeutic treatment and/or promotion of healing of fracture and the like.

TECHNICAL FIELD

The present invention relates to novel nitrogen-containing 6-memberedcyclic compounds and medicaments using them as an active ingredient.

BACKGROUND ART

A bone fracture is a condition in which a bone is partially orcompletely interrupted or deformed by an external force given due toaccident or falling down. Bone fractures are classified into completefracture (break) and incomplete fracture (crack), simple fracture (thereis one fracture line) and comminuted fracture (complicated break ofbone), closed fracture (fracture part is not exposed out of the body)and open fracture (fracture part is exposed out of the body) or thelike. Bone fractures impose serious troubles on patients' daily lifeactivities, and healing thereof takes a considerably long period oftime, although it depends on the fracture part and presence or absenceof slippage (dislocation) of bone. A condition of bone union withoutcorrection of the dislocation is called “malunion”. As conditions ofbone fracture, although it depends on part and type of bone fracture,there may occur such conditions as “delayed union” in which union is notobtained even 3 to 9 months after being wounded due to various factorssuch as aging, diabetes, and smoking, and “nonunion” in which union isnot obtained even 9 months after being wounded, and arrest of thefracture healing process is suspected. In such cases as malunion,delayed union, and nonunion, pain or discomfort accompanies, normalfunctional healing of fracture parts is not obtained, and thereforefracture patients' QOL is markedly degraded.

Considered especially serious problems in the fracture treatment arefractures accompanying osteoporosis. Fractures accompanying osteoporosisfrequently occur at metaphyses of appendicular bones and spine, and inparticular, femoral neck fracture, vertebral compression fracture,fracture of the distal end of radius, and fracture of the proximal endof humerus are regarded as four major fractures observed inosteoporosis. Fractures accompanying osteoporosis have a problem thatredintegration thereof is difficult because of the bone fragility, andeven if osteosynthesis is performed, sufficient stability can hardly beobtained, and inappropriate fixation causes malunion, delayed union, andalso nonunion. Further, since daily activities are restrained during thefracture treatment period, there is induced a negative spiral thatdisuse bone mass reduction and muscular atrophy are advanced, and theynewly invite falling down and fracture. In particular, delay of normalhealing from centrum fracture or femoral neck fracture compels patientsto be bedridden. The incident rates of various and criticalcomplications such as muscular weakness, joint contracture, decubitalulcer, dementia, urinary tract infection, and cardiopulmonaryhypofunction accompanying systemic disuse in these patients areextremely high, and significant reduction of survival rate afterwounding has been reported (Non-patent document 1).

As described above, fractures, especially fractures accompanyingosteoporosis, induce degradation of QOL, severe complications, and alsosignificant influence on vital prognosis, and therefore they poseextremely serious social problems such as increases of health care costand burden of caring. Treatments of fractures are currently performed byreturning the bone condition to that of anatomically normal position,and performing fixation aiming at obtaining healing to the functionallevel before the wounding as far as possible by normal bone restorationprocess mechanism with preventing complications such as malunion,delayed union, and nonunion.

As treatments for positively promoting healing of fractures, ultrasonicfracture treatment apparatuses are used, and as therapeutic drugs, bonemorphogenetic protein (BMP) preparations, parathyroid hormonepreparations, fibroblast growth factor (FGF) preparations and the likeare clinically used, or clinical applications thereof have beenattempted. However, in spite of uses or attempts of use of such varietyof drugs as mentioned above, the number of patients of bone diseasessuch as fracture is ever increasing every year, for example, the numberof femoral neck fracture patients was presumed to be 1,700,000 all overthe world in 1990, and it is predicted to increase to be 6,300,000 in2050. In this respect, development of innovative new drugs havingprophylactic and/or therapeutic effect for bone diseases such asfracture is desired.

It is known that prostaglandin E₂ (henceforth abbreviated as PGE₂) hasvarious physiological functions such as pain-producing action andoxytocic action, and it is also well known that it plays an importantrole in bone metabolism. When PGE₂ is added to a marrow cell culturesystem, the alkaline phosphatase activity, which is a marker ofcalcified bone-like nodule formation and differentiation of osteoblasts,increases. It has been also revealed that when PGE₂ is actuallyadministered to laboratory animals such as rats, or humans, osteogenesisrises, and bone mass increases. Further, when PGE₂ is topicallyadministered to a bone in the form of sustained release preparation,osteogenesis is promoted at the administration site, and thereforeeffect of positively promoting osteogenesis systemically or locally canbe expected for PGE₂.

However, since PGE₂ exhibits side reactions such as pain-producingaction and oxytocic action as described above, which should be avoidedfor continuous long-term administration, there is desired a selectivePGE₂ derivative that safely and effectively acts on bone tissues. Forexample, as the receptors of PGE₂, four kinds of them, EP₁, EP₂, EP₃,and EP₄ receptors, have so far been reported for mouse, rat, dog, humanand the like, and since expression sites thereof and intracellularsignal transduction systems to be activated by them are different,compounds selective for each subtype have been created.

It has been suggested that, among the four kinds of receptors on whichPGE₂ acts, the EP₂ and EP₄ receptors play important roles in bonemetabolism in cells and animals, and both conjugate with the Gs proteinto increase cAMP in osteoblasts. There have been developed EP₂-selectiveagonists, EP₄-selective agonist, and EP₂/EP₄ agonists so far, andsignificant osteogenesis action or fracture healing-promoting effectthereof have been demonstrated in animal models by systemic or localadministration. As compounds that act on the PGE receptors, for example,the compounds described in Patent documents 1 to 8 are known.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: International Patent Publication WO02/24647-   Patent document 2: International Patent Publication WO02/42268-   Patent document 3: International Patent Publication WO03/007941-   Patent document 4: International Patent Publication WO03/035064-   Patent document 5: International Patent Publication WO2004/063158-   Patent document 6: International Patent Publication WO2004/085430-   Patent document 7: U.S. Pat. No. 6,747,037-   Patent document 8: International Patent Publication WO2006/080323

Non-Patent Documents

-   Non-patent document 1: C. Cooper et al., Am. J. Epidemiol., 137,    1001-1005, 1993

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

An object to be achieved by the present invention is to provide a novelcompound having superior EP₄ receptor agonist activity. Preferably, theobject is to provide a novel compound having a superior EP₄receptor-selective agonist activity. Another object is to provide anovel compound useful as an active ingredient of a medicament forprophylactic and/or therapeutic treatment of a disease relating to theEP₄ receptor agonization, for example, a novel compound useful as anactive ingredient of a medicament for therapeutic treatment of fractureand/or promoting healing of fracture. Still another object is to providea medicament containing such a compound.

Means for Achieving the Object

In order to achieve the aforementioned objects, the inventors of thepresent invention conducted various researches. As a result, they foundthat the compounds of the present invention represented by the followingformula (1) have superior EP₄ receptor agonist activity, in particular,the compounds according to a certain embodiment of the present inventionhave superior EP₄ receptor-selective agonist activity, and thosecompounds are useful for prophylactic and/or therapeutic treatment of adisease relating to the EP₄ receptor agonization, for example,therapeutic treatment and/or promotion of healing of fracture, and cameto accomplish the present invention. It is considered that it ispreferable to provide a compound having an EP₄-receptor-selectiveagonist activity for the following reasons. Namely, while the EP₄receptor is observed in osteoblasts and osteoclasts in human culturedosteoblasts and osseous tissues, expression of the EP₂ receptor have notbeen detected (P. Sarrazin, G et al. Prostaglandins Leukot. Essent.Fatty Acids, 64, 203-210, 2001; I. Fortier et al., ProstaglandinsLeukot. Essent. Fatty Acids, 70, 431-439, 2004), and therefore it isconsidered that the most important target of PGE₂ for the action inosseous tissues is the EP₄ receptor, and an EP₄ receptor-selectiveagonist can be a safe and effective medicament for osteoanagenesistreatment. Of course, compounds having an EP₂ receptor agonist activityare not excluded from the compounds of the present invention.

The present invention thus provides the followings.

[1] A compound represented by the following general formula (1);

[wherein, in the formula (1),R¹ represents —H, or halogen;Ar¹ represents any substituent selected from the group which may besubstituted with 1 to 3 of the same or different substituents selectedfrom the group consisting of —F and methyl (provided that

are excluded),wherein the group G¹ is a group consisting of

(a and b represent binding direction);Ar² represents any substituent selected from the group G², which may besubstituted with 1 to 3 of the same or different substituents selectedfrom the group consisting of cyano, —Cl, methyl, methoxy, and phenyl(provided that

are excluded),wherein the group G² is a group consisting of phenyl, thienyl, furyl,and thiazolyl; and* represents an asymmetric carbon],or a salt thereof.[2] The compound or a salt thereof according to [1] mentioned above,wherein R¹ is —H, —Cl, or —Br.[3] The compound or a salt thereof according to [2] mentioned above,wherein Ar¹ is any substituent selected from the group consisting of

[3-2] The compound or a salt thereof according to [1] or [2] mentionedabove, wherein Ar¹ is any substituent selected from the group consistingof

[3-3] The compound or a salt thereof according to [1] or [2] mentionedabove, wherein Ar¹ is any substituent selected from the group consistingof

[4] The compound or a salt thereof according to [2] mentioned above,wherein Ar¹ is

[4-2] The compound or a salt thereof according to [1] or [2] mentionedabove, wherein Ar¹ is

[4-3] The compound or a salt thereof according to [1] or [2] mentionedabove, wherein Ar¹ is

[4-4] The compound or a salt thereof according to [1] or [2] mentionedabove, wherein Ar¹ is

[5] The compound or a salt thereof according to [3] or [4] mentionedabove, wherein Ar² is any substituent selected from the group consistingof

[5-2] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is any substituent selected from thegroup consisting of

When the cited item numbers are indicated with such a range as [1] to[4-4] mentioned above, and the range includes an item indicated with anumber having a subnumber such as [3-2], it is meant that the itemindicated with the number having a subnumber such as [3-2] is alsocited. The same shall apply to the following definitions.

[6] The compound or a salt thereof according to [3] or [4] mentionedabove, wherein Ar² is any substituent selected from the group consistingof

[6-2] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is any substituent selected from thegroup consisting of

[7] The compound or a salt thereof according to [3] or [4] mentionedabove, wherein Ar² is any substituent selected from the group consistingof

[7-2] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is any substituent selected from thegroup consisting of

[7-3] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is any substituent selected from thegroup consisting of

[7-4] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is

[7-5] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is

[7-6] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is

[7-7] The compound or a salt thereof according to any one of [1] to[4-4] mentioned above, wherein Ar² is

[8] The compound or a salt thereof according to [7] mentioned above,wherein R¹ is —H.[8-2] The compound or a salt thereof according to any one of [1] to[7-7] mentioned above, wherein R¹ is —H.[9] The compound or a salt thereof according to [7] mentioned above,wherein R¹ is —Cl.[9-2] The compound or a salt thereof according to any one of [1] to[7-7] mentioned above, wherein R¹ is —Cl.[10] The compound or a salt thereof according to [7] mentioned above,wherein R¹ is —Br.[10-2] The compound or a salt thereof according to any one of [1] to[7-7] mentioned above, wherein R¹ is —Br.[11] The compound or a salt thereof according to [1] mentioned above,wherein R¹ is —H, —Cl, or —Br;Ar¹ is any substituent selected from the group consisting of

andAr² is any substituent selected from the group consisting of

[11-2] The compound or a salt thereof according to [1] mentioned above,wherein R¹ is —H, —Cl, or —Br;Ar¹ is any substituent selected from the group consisting of

andAr² is any substituent selected from the group consisting of

[11-3] The compound or a salt thereof according to [1] mentioned above,wherein R¹ is —H, —Cl, or —Br;Ar¹ is any substituent selected from the group consisting of

andAr² is any substituent selected from the group consisting of

[11-4] The compound or a salt thereof according to [1] mentioned above,wherein R¹ is —H, —Cl, or —Br;Ar¹ is any substituent selected from the group consisting of

andAr² is any substituent selected from the group consisting of

[12] Any compound selected from the following group, or a salt thereof;

[13] The compound mentioned below, or a salt thereof;

[14] The compound mentioned below, or a salt thereof;

[15] The compound mentioned below, or a salt thereof;

[16] The compound mentioned below, or a salt thereof;

[17] The compound mentioned below, or a salt thereof;

[18] The compound mentioned below, or a salt thereof;

[19] The compound mentioned below, or a salt thereof;

[20] A medicament containing the compound according to any one of [1] to[19] mentioned above or a pharmaceutically acceptable salt thereof as anactive ingredient.[21] The medicament according to [20] mentioned above, which is forprophylactic and/or therapeutic treatment of a disease relating to EP₄receptor agonization.[22] The medicament according to [20] mentioned above, which is forpromotion of osteogenesis.[23] The medicament according to [20] mentioned above, which is fortherapeutic treatment and/or promotion of healing of fracture.[24] The medicament according to [20] mentioned above, which is fortherapeutic treatment and/or promotion of healing of bone defect.[25] The medicament according to [20] mentioned above, which is forpromotion of bone union.[25-2] The medicament according to [25] mentioned above, which is forpromotion of bone union in spine fixation.[26] An EP₄ agonist containing the compound according to any one of [1]to [19] mentioned above or a pharmaceutically acceptable salt thereof asan active ingredient.[27] A pharmaceutical composition for therapeutic treatment of fracture,which contains the compound according to any one of [1] to [19]mentioned above or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.[28] A microsphere preparation containing the compound according to anyone of [1] to [19] mentioned above or a pharmaceutically acceptable saltthereof, and a lactic acid/glycolic acid copolymer.[29] The compound according to any one of [1] to [19] mentioned above ora pharmaceutically acceptable salt thereof, which is used fortherapeutic treatment of fracture.[30] A method for therapeutic treatment of fracture in a mammal, whichcomprises the step of administrating an effective amount of the compoundaccording to any one of [1] to [19] mentioned above or apharmaceutically acceptable salt thereof to the mammal.

Effect of the Invention

The “compounds represented by the formula (1) and salts thereof”(henceforth also referred to simply as “the compounds of the presentinvention”) have a superior EP₄ receptor agonist activity. The compoundsof the present invention can be used as an active ingredient of amedicament for prophylactic and/or therapeutic treatment of a diseaserelating to EP₄ receptor agonization, for example, therapeutic treatmentand/or promotion of healing of fracture. As another embodiment, thecompounds of the present invention can be used as a reagent having anEP₄ receptor agonist activity.

MODES FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be specifically explained.

In the present specification, carbon atom may be simply represented as“C”, hydrogen atom as H″, oxygen atom as “O”, sulfur atom as “S”, andnitrogen atom as N″. Further, carbonyl group may be simply representedas “—C(O)—”, carboxyl group as “—COO—”, sulfinyl group as “—S(O)—”,sulfonyl group as “—S(O)₂—”, ether bond as “—O—”, and thioether bond as“—S—” (each “—” in these groups indicates a bond).

In the present specification, the alkyl having 1 to 4 carbon atoms meansmethyl, ethyl, propyl, butyl, or an isomer thereof [normal (n), iso,secondary (sec), tertiary (t) and the like].

In the present specification, the acyl having 2 to 6 carbon atoms meansacetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, or an isomer thereof.

In the present specification, the alkoxy having 1 to 4 carbon atomsmeans methoxy, ethoxy, propoxy, butoxy, or an isomer thereof.

In the present specification, the halogen means fluoro (—F), chloro(—Cl), bromo (—Br), or iodo (—I).

In the present invention, all isomers are included, unless specificallyindicated. For example, the alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylene, alkenylene, and alkynylene include linear and branched groups.Further, any of isomers based on a double bond, ring, or condensed ring(E- or Z-isomers, or cis- or trans-isomers), isomers based on thepresence of an asymmetric carbon and the like (R- or S-isomer, an isomerbased on α- or β-configuration, enantiomers, diastereomers and thelike), optically active substances showing optical rotation (D- orL-isomers, or d- or l-isomers), isomers based on polarity inchromatographic separation (high polarity isomers or low polarityisomers), equilibrated compounds, rotational isomers, mixtures of theseisomers at arbitrary ratios, and racemates fall within the scope of thepresent invention.

In the present specification, as apparent for those skilled in the art,the symbol:

[Formula 39]

indicates that the bond is on the back of the plane (i.e.,α-configuration), the symbol:

[Formula 40]

indicates that the bond is in front of the plane (i.e.,β-configuration), the symbol:

[Formula 41]

means α-configuration or β-configuration, or a mixture thereof, and thesymbol:

[Formula 42]

means a mixture of α-configuration and 6-configuration, unlessespecially indicated.

Hereafter, the compounds represented by the formula (1), or saltsthereof will be explained in detail.

R¹ is, for example, —H, or halogen. According to another embodiment, R¹is, for example, —H, —Cl, or —Br. According to further anotherembodiment, R¹ is, for example, —H.

Ar¹ is, for example, any substituent selected from the group G¹, whichmay be substituted with 1 to 3 of the same or different substituentsselected from the group consisting of —F and methyl (provided that

are excluded), wherein the group G¹ is a group consisting of

(a and b represent binding direction).

According to another embodiment of the group consisting of —F and methylmentioned above, the group consists of, for example, —F, and accordingto further another embodiment, the group consists of, for example,methyl.

According to another embodiment of the group G¹, the group consists of,for example,

According to another embodiment of Ar¹, Ar¹ is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar¹, Ar¹ is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar¹, Ar¹ is, for example,

According to further another embodiment of Ar¹, Ar¹ is, for example,

According to further another embodiment of Ar¹, Ar¹ is, for example,

When Ar¹ is substituted with 1 to 3 of the same or differentsubstituents selected from the group consisting of —F and methyl,according to another embodiment, Ar¹ is, for example, substituted with 1or 2 of the same or different substituents selected from the groupconsisting of —F and methyl, and according to further anotherembodiment, Ar¹ is, for example, substituted with one of —F or methyl.Unsubstituted Ar¹ is also one of preferred embodiments.

Ar² is, for example, any substituent selected from the group G², whichmay be substituted with 1 to 3 of the same or different substituentsselected from the group consisting of cyano, —Cl, methyl, methoxy, andphenyl (provided that

are excluded).

The group G² mentioned above is a group consisting of phenyl, thienyl,furyl, and thiazolyl.

According to another embodiment of the group consisting of cyano, —Cl,methyl, methoxy, and phenyl, the group consists of, for example, cyano.

According to another embodiment of the group G², G² is, for example, agroup consisting of thienyl and furyl.

According to further another embodiment of the group G², the groupconsists of, for example, thienyl.

According to another embodiment of Ar², Ar² is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar², Ar² is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar², Ar² is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar², Ar² is, for example, anysubstituent selected from the group consisting of

According to further another embodiment of Ar², Ar² is, for example,

According to further another embodiment of Ar², Ar² is, for example,

According to further another embodiment of Ar², Ar² is, for example,

According to further another embodiment of Ar², Ar² is, for example,

When Ar² is substituted with 1 to 3 of the same or differentsubstituents selected from the group consisting of cyano, —Cl, methyl,methoxy, and phenyl, according to another embodiment, Ar² is, forexample, substituted with 1 or 2 of the same or different substituentsselected from the group consisting of cyano, —Cl, methyl, methoxy, andphenyl, and according to further another embodiment, Ar² is, forexample, substituted with 1 of cyano, —Cl, methyl, methoxy, or phenyl.Unsubstituted Ar² is also one of preferred embodiments.

As specific compounds falling within the scope of the present invention,the following compounds can be exemplified.

As further other specific examples of the compounds falling within thescope of the present invention, the following compounds can beexemplified.

In this specification, the “compounds represented by the formula (1)” isgenerally understood as the compounds represented by the formula (1) inthe free form. Examples of the salt thereof include the following salts.

The type of the salt of the compounds represented by the formula (1) isnot particularly limited, and it may be an acid addition salt, or a baseaddition salt, and may be in the form of an intramolecular counter ion.In particular, when the salt is used as an active ingredient of amedicament, the salt is preferably a pharmaceutically acceptable salt.When disclosure is made for use as a medicament in this specification,the salt of the compounds represented by the formula (1) is usuallyunderstood as a pharmaceutically acceptable salt. Acid addition saltsinclude, for example, acid addition salts with an inorganic acid such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,nitric acid, and phosphoric acid, and acid addition salts with anorganic acid such as formic acid, acetic acid, propionic acid, oxalicacid, malonic acid, succinic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, citric acid, malic acid, tartaric acid,dibenzoyltartaric acid, mandelic acid, maleic acid, fumaric acid,aspartic acid, and glutamic acid. As base addition salts, for example,base addition salts with an inorganic base such as sodium, potassium,magnesium, calcium, and aluminum, base addition salts with an organicbase such as methylamine, 2-aminoethanol, arginine, lysine, andornithine and the like can be exemplified. However, the type of the saltis not limited to these, and it can of course be appropriately selectedby those skilled in the art.

The compounds of the present invention may be in the form of hydrate.The compounds of the present invention may also be in the form ofanhydride.

The compounds of the present invention may be in the form of solvate.The compounds of the present invention may also be in the form ofnon-solvate.

The compounds of the present invention may be in the form of crystal.The compounds of the present invention may also be in an amorphous form.

More specifically, the compounds of the present invention includeanhydrides and non-solvates of the “compounds represented by the formula(1)”, hydrates and/or solvates thereof, and crystals thereof.

The compounds of the present invention also include anhydrides andnon-solvates of “salts of the compounds represented by the formula (1)”,hydrates and/or solvates of the salts, and crystals thereof.

The compounds of the present invention may also be a pharmaceuticallyacceptable prodrug of “the compounds represented by the formula (1)”.The pharmaceutically acceptable prodrug is a compound having a groupthat can be changed into amino group, hydroxyl group, carboxyl group orthe like by solvolysis or under physiological conditions. For example,as a group that forms a prodrug for hydroxy group, or amino group, forexample, an acyl group and an alkoxycarbonyl group are exemplified. As agroup that forms a prodrug for carboxyl group, for example, methylgroup, ethyl group, n-propyl group, isopropyl group, n-butyl group,isobutyl group, s-butyl group, t-butyl group, amino group, methylaminogroup, ethylamino group, dimethylamino group, and diethylamino group areexemplified.

Such a prodrug can be prepared by, for example, appropriatelyintroducing a group that forms a prodrug into any of the compounds ofthe present invention at one or more arbitrary groups selected fromhydroxyl group and amino group using a prodrug-forming reagent such as acorresponding halide in a conventional manner, then, if desired,appropriately isolating and purifying the compound in a conventionalmanner. A group that forms a prodrug can also be appropriatelyintroduced into the compound of the present invention at carboxyl groupby using such a prodrug-forming reagent as a corresponding alcohol oramine in a conventional manner.

The compounds of the present invention may have an asymmetric carbon.The steric configuration of such an asymmetric carbon is notparticularly limited, and it may be in the S-configuration orR-configuration, or a mixture of the both. Any optically activesubstances based on such an asymmetric carbon in a pure form,stereoisomers such as diastereoisomers, arbitrary mixtures ofstereoisomers, racemates and the like all fall within the scope of thecompounds of the present invention.

In particular, the steric configuration of the asymmetric carbonindicated with “*” in the formula (1) is not particularly limited.However, the configuration shown below is one of the preferredembodiments.

When Ar¹ is benzene ring among the groups of the group G¹, the stericconfiguration in the above formula is the S-configuration, and when Ar¹is thiophene ring among the groups of the group G¹, the stericconfiguration in the above formula is the R-configuration.

<Preparation Methods of the Compounds of the Present Invention>

The compounds of the present invention are novel compounds not describedin literature. Although the compounds of the present invention can beprepared by, for example, the following methods, the preparation methodof the compounds of the present invention is not limited to thefollowing methods.

Although reaction time in each of the reactions is not particularlylimited, progress of the reactions can be easily monitored by analysismethods described later, and therefore the reactions may be terminatedwhen the maximum yield of objective substance is obtained. Each of thereactions can be performed in an inert gas atmosphere, for example,under a nitrogen flow or an argon flow, as required. When protectionwith a protective group and subsequent deprotection are needed in eachof the reactions, the reactions can be appropriately performed byutilizing the methods described below.

Examples of the protective group used in the present invention includethe following groups: protective groups for carboxyl group (—COOH),protective groups for hydroxyl group (—OH), protective groups for analkynyl group, protective groups for amino group (—NH₂) and the like.

Examples of the protective group for carboxyl group include, forexample, an alkyl having 1 to 4 carbon atoms, an alkenyl having 2 to 4carbon atoms, an alkyl having 1 to 4 carbon atoms and substituted withan alkoxy having 1 to 4 carbon atoms, an alkyl having 1 to 4 carbonatoms and substituted with 1 to 3 halogens and the like. Specificexamples include methyl, ethyl, t-butyl, allyl, methoxyethyl,trichloroethyl and the like.

Examples of the protective groups for hydroxyl group include, forexample, an alkyl having 1 to 4 carbon atoms, an alkenyl having 2 to 4carbon atoms, an alkyl having 1 to 4 carbon atoms and substituted withan alkoxy having 1 to 4 carbon atoms, an alkyl having 1 to 4 carbonatoms and substituted with 1 to 3 halogens, a silyl substituted withthree of the same or different alkyls having 1 to 4 carbon atoms orphenyls, tetrahydropyranyl, tetrahydrofuryl, propargyl,trimethylsilylethyl group and the like. Specific examples includemethyl, ethyl, t-butyl, allyl, methoxymethyl (MOM), methoxyethyl (MEM),trichloroethyl, phenyl, methylphenyl, chlorophenyl, benzyl,methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl,trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, N-methylaminobenzyl,N,N-dimethylaminobenzyl, phenacyl, trityl, 1-ethoxyethyl (EE),tetrahydropyranyl (THP), tetrahydrofuryl, propargyl, trimethylsilyl(TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS),t-butyldiphenylsilyl(TBDPS), acetyl (Ac), pivaloyl, benzoyl,allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc) and thelike.

Examples of the protective groups for alkynyl include trimethylsilyl,2-hydroxy-2-propyl and the like.

Examples of the protective groups for amino group include, for example,benzyl, methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl,trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, N-methylaminobenzyl,N,N-dimethylaminobenzyl, phenacyl, acetyl group, trifluoroacetyl,pivaloyl, benzoyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,benzyloxycarbonyl, t-butoxycarbonyl (Boc),1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc),9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM),2-(trimethylsilyl)ethoxymethyl (SEM) and the like.

By removing these protective groups simultaneously with the preparationor stepwise during the preparation process or at the final step,protected compounds can be converted into objective compounds. Theprotection and deprotection reactions can be performed according toknown methods such as the methods described in, for example, ProtectiveGroups in Organic Synthesis, published by John Wiley and Sons (2007) andthe like, and they can be performed by, for example, the methods of (1)to (6) mentioned below and the like.

(1) The deprotection reaction by alkali hydrolysis is performed by, forexample, reacting a protected compound with a base in a polar solvent.Examples of the base used in this reaction include, for example, alkalimetal bases such as sodium hydroxide, potassium hydroxide, lithiumhydroxide, barium hydroxide, calcium hydroxide, sodium carbonate,potassium carbonate, sodium methoxide, and potassium t-butoxide, andorganic bases such as triethylamine. For example, they are usually usedin an amount of 1 to 20 fold moles, preferably 1 to 10 fold moles, basedon the reactant, when an alkali metal base is used, or 1 fold mole to alarge excess amount, when an organic base is used. As for the reactionsolvent, it is usually preferred that the reaction is performed in aninactive medium that does not inhibit the reaction, preferably a polarsolvent. Examples of the polar solvent include water, methanol, ethanol,tetrahydrofuran, dioxane and the like, and these can be used as amixture as required. As the reaction temperature, a suitabletemperature, for example, from −10° C. to the reflux temperature of thesolvent, is chosen. The reaction time is, for example, usually 0.5 to 72hours, preferably 1 to 48 hours, when an alkali metal base is used, orusually 5 hours to 14 days, when an organic base is used. However, theprogress of the reaction can be monitored by thin layer chromatography(TLC), high performance liquid chromatography (HPLC) or the like, andaccordingly, the reaction may usually be terminated when the maximumyield of the objective compound is obtained.(2) The deprotection reaction under an acidic condition is performed,for example, in an organic solvent (dichloromethane, chloroform,dioxane, ethyl acetate, anisole and the like) in the presence of anorganic acid (acetic acid, trifluoroacetic acid, methanesulfonic acid,p-toluenesulfonic acid and the like), a Lewis acid (boron tribromide,boron trifluoride, aluminum bromide, aluminum chloride and the like), oran inorganic acid (hydrochloric acid, sulfuric acid and the like), or amixture thereof (hydrogen bromide/acetic acid and the like) at atemperature of −10 to 100° C. There is also a method of addingethanethiol, 1,2-ethanedithiol or the like as an additive.(3) The deprotection reaction by hydrogenolysis is performed, forexample, in a solvent [ether type solvents (tetrahydrofuran, dioxane,dimethoxyethane, diethyl ether and the like), alcohol type solvents(methanol, ethanol and the like), benzene type solvents (benzene,toluene and the like), ketone type solvents (acetone, methyl ethylketone and the like), nitrile type solvents (acetonitrile and the like),amide type solvents (dimethylformamide and the like), ester typesolvents (ethyl acetate and the like), water, acetic acid, mixtures oftwo or more types of those solvents and the like] in the presence of acatalyst (palladium/carbon powder, platinum oxide (PtO₂), activatednickel and the like) and a hydrogen source such as hydrogen gas ofordinary pressure or under pressurization, ammonium formate, orhydrazine hydrate at a temperature of −10 to 60° C.(4) The deprotection reaction of silyl group is performed, for example,by using tetra-n-butylammonium fluoride or the like in a water-miscibleorganic solvent (tetrahydrofuran, acetonitrile and the like) at atemperature of −10 to 60° C.(5) The deprotection reaction using a metal is performed, for example,in an acidic solvent (acetic acid, buffer of pH 4.2 to 7.2, a mixture ofsuch a solution and an organic solvent such as tetrahydrofuran) in thepresence of zinc powder with or without ultrasonication at a temperatureof −10 to 60° C.(6) The deprotection reaction using a metal complex is performed, forexample, in an organic solvent (dichloromethane, dimethylformamide,tetrahydrofuran, ethyl acetate, acetonitrile, dioxane, ethanol and thelike), water, or a mixture thereof in the presence of a trap reagent(tributyltin hydride, triethylsilane, dimedone, morpholine,diethylamine, pyrrolidine and the like), an organic acid (acetic acid,formic acid, 2-ethylhexanoic acid and the like) and/or an organic acidsalt (sodium 2-ethylhexanoate, potassium 2-ethylhexanoate and the like)in the presence or absence of a phosphine type regent(triphenylphosphine and the like) by using a metal complex[tetrakistriphenylphosphine palladium(0),bis(triphenylphosphine)palladium(II) dichloride, palladium(II) acetate,tris(triphenylphosphine) rhodium(I) chloride and the like] at atemperature of −10 to 60° C.

The compounds of the present invention represented by the formula (1)can be prepared, for example, according to the following reactionpathways. In the following schemes, “STEP” means each step, for example,“STEP 1-1” means Step 1-1.

Step 1-1

The compounds represented by the formula (1) can be prepared bydeprotection of a compound represented by the formula (2) [in theformula (2), “Pro” represents a protective group of carboxyl in theformula (1)] for the protective group Pro¹. The deprotection reactioncan be carried out according to known methods, for example, the methodsdescribed in Protective Groups in Organic Synthesis, published by JohnWiley and Sons (2007) and the like.

Pro¹ is not particularly limited so long as it is the protective groupof carboxyl mentioned above, and examples thereof include, for example,an alkyl having 1 to 4 carbon atoms.

Step 1-2

The compounds represented by the formula (2) can be prepared bydeprotection of a compound represented by the formula (3) [in theformula (3), “Pro²” represents a protective group of hydroxyl group inthe formula (1), and “Pro¹” has the same meaning as that defined above]for the protective group of the compound represented by the formula (3).The deprotection reaction can be carried out according to known methods,for example, the methods described in Protective Groups in OrganicSynthesis, published by John Wiley and Sons (2007) and the like.

Although Pro² is not particularly limited so long as it is theaforementioned protective group of hydroxyl group, Pro² is preferably agroup other than TMS in order to selectively perform deprotection forPro² with respect to TMS in the formula (5). Examples of Pro² include,for example, tert-butyl group, MOM group, MEM group, THP group, acetylgroup, and TBDMS group.

Step 1-3

The compounds represented by the formula (3) can be prepared by couplinga compound represented by the formula (4) [in the formula (4), “Pro¹”and “Pro²” have the same meanings as those defined above], and acompound represented by the formula (11) [in the formula (11), “hal¹”represents bromo or iodo] in the presence of a base and a palladiumcatalyst. As for the amount of the compound represented by the formula(11) used in the reaction of the compound represented by the formula (4)and the compound represented by the formula (11), ⅕ to 20 equivalents,preferably ½ to 10 equivalents, more preferably 1 to 5 equivalents ofthe compound represented by the formula (11) can be used with respect tothe compound represented by the formula (4). However, the amount of thecompound represented by the formula (11) to be used can be appropriatelydetermined in consideration of purity, yield, purification efficiencyand the like of the compound represented by the formula (4).

As the base, for example, cesium carbonate, sodium carbonate, potassiumcarbonate and the like can be used, and cesium carbonate is preferred.As for the amount of the base to be used, it can be used in an amount offrom equivalent amount to excess amount, for example, 1 to 10equivalents, preferably 1 to 5 equivalents, with respect to the compoundrepresented by the formula (4), which serves as the starting material.

As the palladium catalyst, for example, marketed catalysts such astetrakis(triphenylphosphine)palladium,tetrakis(methyldiphenylphosphine)palladium,dichlorobis(triphenylphosphine)palladium,dichlorobis(tri-o-tolylphosphine)palladium,dichlorobis(tricyclohexylphosphine)palladium,dichlorobis(triethylphosphine)-palladium, palladium acetate, palladiumchloride, bis(acetonitrile)palladium chloride,bis(dibenzylideneacetone)palladium,tris(dibenzylideneacetone)dipalladium, andbis(diphenylphosphinoferrocene)palladium chloride may be added to thereaction system as they are, or a catalyst separately prepared frompalladium acetate, tris(dibenzylideneacetone)dipalladium or the like,and an arbitrary ligand, and isolated may be added. A catalystconsidered to actually participate in the reaction may be prepared inthe reaction system by mixing palladium acetate,tris(dibenzylidene-acetone)dipalladium or the like, and an arbitraryligand. The valence of palladium may be 0 or +2. In particular,bis(acetonitrile)palladium chloride can be mentioned as a preferredexample.

As the ligand used for preparing a palladium catalyst from an arbitraryligand, there are exemplified phosphine ligands such astriphenylphosphine, tri(o-tolyl)-phosphine, tri(cyclohexyl)phosphine,tri(t-butyl)phosphine, dicyclohexyl-phenylphosphine,1,1′-bis(di-t-butylphosphino)ferrocene,2-dicyclohexylphosphino-2′-dimethylamino-1,1′-biphenyl,2-(di-t-butylphosphino)biphenyl, 2-(dicyclohexyl-phosphino)biphenyl,2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, xantphos, andtri(tert-butyl)phosphine. There are also exemplified2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl,2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl,1,2,3,4,5-pentamethyl-1′-(di-t-butylphosphino)ferrocene) and the like,and 2-dicyclohexyl-2′,4′,6′-triisopropylbiphenyl can be preferablymentioned.

Although the amount in equivalence of the palladium catalyst to be usedmay be an equivalent amount or catalytic amount, it is preferably 0.01mol % or more, more preferably, especially 0.10 to 50.0 mol %, based onthe amount of the starting compound. Examples of the solvent used forthe reaction include, for example, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene, toluene, 1,4-dioxane, andtetrahydrofuran, and preferred examples include acetonitrile. Two ormore kinds of these solvents can also be used as a mixture. As for thereaction temperature, the reaction can be performed usually at −40 to100° C., preferably at −20° C. to 60° C. Although the reaction time isnot particularly limited, it is, for example, usually 0.5 to 48 hours,preferably 1 to 24 hours.

Step 1-4

The compounds represented by the formula (4) can be prepared byselective deprotection of TMD of a compound represented by the formula(5) [in the formula (5), “Pro¹” and “Pro²” have the same meanings asthose defined above]. The deprotection reaction can be carried outaccording to known methods, for example, the methods described inProtective Groups in Organic Synthesis, published by John Wiley and Sons(2007) and the like.

Specifically, the compounds can be prepared by, for example, allowing aninorganic base to act on a compound represented by the formula (5) in anorganic solvent. As the inorganic base, for example, sodium hydroxide,potassium hydroxide, cesium carbonate, sodium carbonate, potassiumcarbonate or the like can be used, and potassium carbonate is preferred.As for the amount of the base to be used, the base can be used in anamount of from equivalent amount to excess amount with respect to thecompound represented by the formula (5), which serves as a startingmaterial, and the amount is, for example, 1 to 10 equivalents,preferably 1 to 5 equivalents. Examples of the solvent used for thereaction include methanol and ethanol, and preferred examples includemethanol. As for the reaction temperature, the reaction can be performedusually at −20 to 60° C., preferably at 0 to 40° C. Although thereaction time is not particularly limited, it is, for example, usually0.5 to 48 hours, preferably 1 to 24 hours.

Step 1-5

The compounds represented by the formula (5) can be prepared by couplinga compound represented by the formula (6) [in the formula (6), “Pro¹”and “Pro²” have the same meanings as those defined above, and “hal²”represents bromo or iodo] with a compound represented by the formula(13) in an organic solvent in the presence of an inorganic base. Thecompounds can be prepared in the same manner as that of the Step 1-3. Inthis preparation, the compound represented by the formula (13) can beused in an amount of ⅕ to 20 equivalents, preferably ½ to 10equivalents, more preferably 1 to 5 equivalents, with respect to thecompound represented by the formula (6).

Step 1-6

The compounds represented by the formula (6) can be prepared byprotecting the hydroxyl group of a compound represented by the formula(7) [in the formula (7), “Pro¹” and “hal²” have the same meanings asthose defined above]. The protection reaction for hydroxyl group can becarried out according to known methods, for example, the methodsdescribed in Protective Groups in Organic Synthesis, published by JohnWiley and Sons (2007) and the like. Although the protective group ofhydroxyl group is not particularly limited so long as the aforementionedprotective group of hydroxyl group is chosen, for example, tert-butylgroup, MOM group, MEM group, THP group, acetyl group, TBDMS group andthe like can be used.

Step 1-7

The compounds represented by the formula (7) can be prepared by allowinga reducing agent to act on a compound represented by the formula (9) [inthe formula (9), “Pro¹” and “hal²” have the same meanings as thosedefined above] in an organic solvent. As the reducing agent, forexample, sodium borohydride, lithium borohydride, triacetoxyborohydride,cyanoborohydride and the like can be used, and sodium borohydride ispreferred. As for the amount of the reducing agent to be used, it can beused in an amount of ¼ equivalent to excess amount with respect to thecompound represented by the formula (9), which serves as the startingmaterial, and the amount is, for example, ¼ to 10 equivalents,preferably 1 to 5 equivalents. Examples of the organic solvent used forthe reaction include methanol, ethanol, isopropanol, and a mixed solventof these with tetrahydrofuran, and preferred examples include methanol.As for the reaction temperature, the reaction can be performed usuallyat −20 to 60° C., preferably at 0 to 40° C. Although the reaction timeis not particularly limited, it is, for example, usually 0.5 to 48hours, preferably 1 to 24 hours.

Step 1-8

The compounds represented by the formula (9) can be prepared by allowinga compound represented by the formula (14) [in the formula (14), “hale”has the same meaning as that defined above] to act on a compoundrepresented by the formula (10) [in the formula (10), “Pro¹” has thesame meaning as that defined above]. As for the amount of the compoundrepresented by the formula (14) to be used, it can be used in an amountof from equivalent amount to excess amount with respect to the compoundrepresented by the formula (10), which serves as the starting material,and the amount is, for example, equivalent amount to 10 equivalents,preferably 1 to 5 equivalents. Examples of the solvent used for thereaction include methanol, ethanol, isopropanol, and a mixed solvent ofthese with water, and preferred examples include ethanol. As for thereaction temperature, the reaction can be performed usually at 0 to 120°C., preferably at 40 to 100° C. Although the reaction time is notparticularly limited, it is, for example, usually 0.5 to 48 hours,preferably 1 to 24 hours.

Step 1-9

The compounds represented by the formula (3) may also be prepared from acompound represented by the formula (6) [in the formula (6), “Pro¹”,“Pro²”, and “hal²” have the same meanings as those defined above], and acompound represented by the general formula (12) in the same manner asthat of the method of Step 1-3. As for the amount of the compoundrepresented by the formula (12) used in this case, it can be used in anamount of ⅕ to 20 equivalents, preferably ½ to 10 equivalents, morepreferably 1 to 5 equivalents, with respect to the compound representedby the formula (6).

Step 1-10

The compounds represented by the formula (2) may also be prepared from acompound represented by the formula (8) [in the formula (8), “Pro¹” hasthe same meaning as that defined above] in the same manner as the methodof Step 1-7.

Step 1-11

The compounds represented by the formula (8) are prepared by coupling acompound represented by the formula (9) wherein “hal²” is iodine atom[in the formula (9), “Pro¹” has the same meaning as that defined above],and a compound represented by the formula (12) in the presence of abase, a copper catalyst, and a palladium catalyst. As for the amount ofthe compound represented by the formula (12) used in the reaction of thecompound represented by the formula (9), and the compound represented bythe formula (12), it can be used in an amount of ⅕ to 20 equivalents,preferably ½ to 10 equivalents, more preferably 1 to 5 equivalents, withrespect to the compound represented by the formula (9), but it may beappropriately determined in consideration of purity, yield, purificationefficiency and the like of the compound represented by the formula (8).

As the base, for example, triethylamine, diethylamine, diisopropylamine,diisopropylethylamine, morpholine, piperidine, pyridine and the like canbe used, and diester amines are preferred. As for the amount of the baseto be used, it can be used in an amount of from equivalent amount toexcess amount, for example, 1 to 10 equivalents, preferably 1 to 5equivalents, with respect to the compound represented by the formula(9), which serves as the starting material.

Examples of the copper catalyst include, for example, copper(I) iodide,copper(I) bromide, copper(I) chloride and the like, and copper(I) iodideis preferred.

Although the amount in equivalence of the copper catalyst to be used maybe an equivalent amount or catalytic amount, it is preferably 0.01 mol %or more, particularly preferably 0.10 to 50.0 mol %, based on thestarting material compound.

As the palladium catalyst, for example, marketed catalysts such astetrakis(triphenylphosphine)palladium,tetrakis(methyldiphenylphosphine)palladium,dichlorobis(triphenylphosphine)palladium,dichlorobis(tri-o-tolylphosphine)palladium,dichlorobis(tricyclohexylphosphine)palladium,dichlorobis(triethylphosphine)-palladium, palladium acetate, palladiumchloride, bis(acetonitrile)palladium chloride,bis(dibenzylideneacetone)palladium,tris(dibenzylideneacetone)dipalladium, andbis(diphenylphosphinoferrocene)palladium chloride may be added to thereaction system as they are, or a catalyst separately prepared frompalladium acetate, tris(dibenzylideneacetone)dipalladium or the like,and an arbitrary ligand and isolated may be added. A catalyst consideredto actually participate in the reaction may be prepared in the reactionsystem by mixing palladium acetate,tris(dibenzylidene-acetone)dipalladium or the like, and an arbitraryligand. The valence of palladium may be 0 or +2. In particular,tetrakis(triphenylphosphine)palladium can be mentioned as a preferredexample. When the palladium catalyst is prepared from an arbitraryligand, the same ligands as the ligands exemplified for Step 1-3 can beused.

Although the amount in equivalence of the palladium catalyst to be usedmay be equivalent amount or a catalytic amount, it is preferably 0.01mol % or more, more preferably, especially 0.10 to 50.0 mol %, based onthe starting material compound.

Examples of the solvent used for the reaction include, for example,N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene,toluene, 1,4-dioxane, tetrahydrofuran and the like, or the reaction canalso be performed without solvent. The reaction performed withoutsolvent is a preferred example. As for the reaction temperature, thereaction can be performed usually at −40 to 100° C., preferably at −20to 60° C. Although the reaction time is not particularly limited, it is,for example, usually 0.5 to 48 hours, preferably 1 to 24 hours.

Step 1-12

The compounds represented by the formula (8) may also be prepared from acompound represented by the formula (10) [in the formula (10), “Pro¹”has the same meaning as that defined above], and a compound representedby the formula (15) in the same manner as that of Step 1-8.

Step 2-1

The compounds represented by the formula (10) can be prepared byallowing a base to act on a compound represented by the formula (A1) [inthe formula (A1), “Pro¹” has the same meaning as that defined above] inan organic solvent. As the base, for example, sodium hydroxide,potassium hydroxide, cesium carbonate, sodium carbonate, sodiumhydrogencarbonate, potassium carbonate and the like can be used, andsodium hydrogencarbonate is preferred. The base can be used in an amountof equivalent amount to an excess amount, for example, 1 to 20equivalents, preferably 1 to 10 equivalents, with respect to thecompound represented by the formula (10), which serves as the startingmaterial. Sodium iodide can be used as an additive, and can be used inan amount of equivalent amount to an excess amount, for example, 1 to 10equivalents, preferably 1 to 5 equivalents, with respect to the compoundrepresented by the formula (10), which serves as the starting material.Examples of the organic solvent used for the reaction includeN,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, toluene,tetrahydrofuran, 1,4-dioxane, diethyl ether, and mixed solvents ofthese, and preferred examples include acetonitrile. As for the reactiontemperature, the reaction can be performed usually at 0 to 100° C.,preferably at 20 to 60° C. Although the reaction time is notparticularly limited, it is, for example, usually 0.5 to 48 hours,preferably 1 to 24 hours.

Step 2-2

The compounds represented by the formula (A1) can be prepared bydeprotection of a compound represented by the formula (A2) [in theformula (A2), “Pro” has the same meaning as that defined above] for theprotective group. The deprotection reaction can be carried out accordingto known methods, for example, the methods described in ProtectiveGroups in Organic Synthesis, published by John Wiley and Sons (2007) andthe like.

Step 2-3

The compounds represented by the formula (A2) can be prepared byallowing a chlorothioformic acid ester such as 2-chloroethylchlorothioformate to act on a compound represented by the formula (A3)[in the formula (A3), “Pro” has the same meaning as that defined above]in the presence of a base. The chlorothioformic acid ester can be usedin an amount of from equivalent amount to excess amount, for example, 1to 5 equivalents, preferably 1 to 2 equivalents, with respect to thecompound represented by the formula (A2), which serves as the startingmaterial. As the base to be used, for example, sodium carbonate,potassium carbonate, sodium hydrogencarbonate, cesium carbonate, sodiumhydroxide, diisopropylethylamine, triethylamine and the like can beused, and sodium hydrogencarbonate is preferred. Examples of the solventused for the reaction include dichloromethane, toluene, tetrahydrofuran,1,4-dioxane, acetonitrile and the like, and dichloromethane ispreferred. As for the reaction temperature, the reaction can beperformed at 0 to 100° C., preferably at 10 to 30° C. Although thereaction time is not particularly limited, it is, for example, usually 1to 24 hours, preferably 2 to 4 hours.

Step 2-4

The compounds represented by the formula (A3) can be prepared byallowing t-butoxycarbonylhydrazine to act on a compound represented bythe formula (A4) [in the formula (A4), “Pro¹” has the same meanings asthat defined above] in the presence of a base. As the base to be used,for example, sodium carbonate, potassium carbonate, sodiumhydrogencarbonate, cesium carbonate, sodium hydroxide,diisopropylethylamine, triethylamine and the like can be used, andsodium hydrogencarbonate is preferred. The amount of the base to be usedis, for example, 1 to 20 equivalents, preferably 3 to 5 equivalents,with respect to the compound represented by the formula (A4), whichserves as the starting material. Sodium iodide or the like can be usedas an additive. Examples of the solvent used for the reaction includeacetonitrile, propionitrile, N,N-dimethylformamide, dimethyl sulfoxide,N-methylpyrrolidone and the like can be used, and preferred examplesinclude acetonitrile. As for the reaction temperature, the reaction canbe performed usually at room temperature to 150° C., preferably at 70°C. to 100° C. Although the reaction time is not particularly limited, itis, for example, 3 to 36 hours, preferably 3 to 18 hours.

Step 2-5

The compounds represented by the formula (A4) can be prepared bysubstituting bromine atom for the hydroxyl group of a compoundrepresented by the formula (A5) [in the formula (A5), “Pro¹” has thesame meaning as that defined above]. The reaction for substitutingbromine atom can be performed by allowing carbon tetrabromide,N-bromosuccinimide or the like to act on the compound in the presence oftriphenylphosphine or the like. The amount of triphenylphosphine to beused is, for example, 1 to 5 equivalents, preferably 1 to 2 equivalents,with respect to the compound represented by the formula (A5), which isthe starting material. The amount of carbon tetrabromide or the like tobe used is, for example, 1 to 5 equivalents, preferably 1 to 2equivalents, with respect to the compound represented by the formula(A5), which is the starting material. Examples of the solvent used forthe reaction include dichloromethane, toluene, tetrahydrofuran,1,4-dioxane, acetonitrile and the like, and dichloromethane ispreferred. As for the reaction temperature, the reaction can beperformed usually at −20 to 40° C., preferably at −10 to 10° C. Althoughthe reaction time is not particularly limited, it is, for example,usually 3 to 36 hours, preferably 12 to 20 hours.

Step 2-6

The compounds represented by the formula (A5) can be prepared bydeprotection of a compound represented by the formula (6A) for theprotective group of the hydroxyl group. The deprotection can be carriedout according to known methods, for example, the methods described inProtective Groups in Organic Synthesis, published by John Wiley and Sons(2007) and the like.

Step 2-6

The compounds represented by the formula (A5) can be prepared byconverting the carboxylic acid of a compound represented by the formula(A6) into ester, and performing deprotection to remove the protectivegroup of the hydroxyl group. The reaction can be advanced in an alcoholsolvent in the presence of an acid. Examples of the acid used for thereaction include sulfuric acid, hydrogen chloride, methanesulfonic acid,p-toluenesulfonic acid, trifluoroacetic acid and the like, and preferredexamples include sulfuric acid. As the solvent, methanol, ethanol or thelike can be used, and preferred examples include methanol. As for thereaction temperature, the reaction can be performed usually at roomtemperature to 140° C., preferably at 50 to 80° C. Although the reactiontime is not particularly limited, it is, for example, usually 2 to 24hours, preferably 8 to 16 hours.

Step 2-7

The compounds represented by the formula (A6) can be prepared byallowing a strong base, and then carbon dioxide to act on a compoundrepresented by the formula (A7). As the strong base, lithium amide suchas diisopropyl lithium amide or lithium hexamethyl disilazide can beused. When R¹ is hydrogen, a lower alkyllithium such as n-butyllithium,s-butyllithium, and n-propyllithium can also be used, and diisopropyllithium amide is preferably used. The amount of the strong base used is,for example, 1 to 3 equivalents, preferably 1 to 2 equivalents, withrespect to the compound represented by the formula (A7), which is thestarting material. Examples of the solvent used for the reaction includetetrahydrofuran, diethyl ether, 1,4-dioxane and the like, andtetrahydrofuran is preferred. As for the temperature for the reactionwith the strong base, the reaction can be performed usually at −100 to−20° C., preferably at −80 to −60° C. The following reaction with carbondioxide or the like can usually be performed at −40 to 40° C., andpreferably −20 to 10° C. Although the reaction time of the reaction withthe strong base is not particularly limited, it is, for example, 0.2 to3 hour, preferably 0.5 to 1 hours. Although the reaction time of thereaction with carbon dioxide or the like is not particularly limited, itis, for example, usually 0.5 to 24 hours, preferably 0.75 to 2 hours.

Step 2-8

The compounds represented by the formula (A7) can be prepared byprotecting the hydroxyl group of a compound represented by the formula(A8) with TBDMS. The protection of the hydroxyl group can be performedby using a method similar to that of Step 1-6.

The compound of the formula (A8) wherein R¹ is H is a marketed compound(2-(thiophen-2-yl)ethanol, Tokyo Chemicals). Therefore, when R¹ in theformula (A8) is H, the following steps are not required.

Step 2-9

The compounds represented by the formula (A8) can be prepared byreducing the ester group of a compound represented by the formula (A9)[in the formula (A9), “Pro³” represents a protective group of thecarboxyl of the compound of the formula (A8)]. That is, as Pro³, forexample, alkyl having 1 to 4 carbon atoms can be used.

As the reducing agent, for example, lithium aluminum hydride,diisobutylaluminum hydride, lithium hydride-triethylborane and the likecan be used, and lithium aluminum hydride is preferred. The amount ofthe reducing agent to be used is, for example, 0.5 to 5 mol equivalents,preferably 1 to 2 mol equivalents, with respect to the compoundrepresented by the formula (A9), which serves as the starting material.

Examples of the solvent used for the reaction include tetrahydrofuran,diethyl ether, toluene, and mixed solvents of these, and preferredexamples include tetrahydrofuran and diethyl ether. As for the reactiontemperature, the reaction can be performed usually at −10 to 20° C.,preferably at −5 to 5° C. Although the reaction time is not particularlylimited, it is, for example, usually 0.08 to 0.5 hour, preferably 0.15to 0.3 hour.

Step 2-10

The compounds represented by the formula (A9) can be prepare by, forexample, carrying out solvolysis of a compound represented by theformula (A10) in an alcohol in the presence of an acid. As the acid,sulfuric acid, methanesulfonic acid, hydrogen chloride and the like canbe used, and sulfuric acid is preferred. The amount of sulfuric acid tobe used is, for example, 0.0001 to 0.005 mol equivalent, preferably0.0002 to 0.001 mol equivalent, with respect to the compound representedby the formula (A10), which serves as the starting material. As thealcohol used as the solvent, for example, ethanol, methanol, n-propanol,n-butyl alcohol, isobutyl alcohol and the like can be used. Although thereaction time is not particularly limited, it is, for example, usually 6to 48 hours, preferably 16 to 24 hours.

Step 2-11

The compounds represented by the formula (A10) can be prepared byallowing a cyanide to act on a compound represented by the formula(A11). As the cyanide, for example, sodium cyanide, potassium cyanideand the like can be used. The amount of the cyanide to be used is, forexample, 1 to 5 equivalents, preferably 1 to 2 equivalents, with respectto the compound represented by the formula (A10), which is the startingmaterial. As the solvent used for the reaction, tetrahydrofuran,acetonitrile, dimethyl sulfoxide, N,N-dimethylacetamide,N,N-dimethylformamide and the like can be used, and preferred examplesinclude a mixed solvent of acetonitrile and dimethyl sulfoxide. As forthe reaction temperature, the reaction can be performed usually at 0 to60° C., preferably 10 to 40° C. Although the reaction time is notparticularly limited, it is, for example, usually 0.5 to 20 hours,preferably 2 to 6 hours.

Step 2-12

The compounds represented by the formula (A11) can be prepared byconverting the hydroxyl group of a compound represented by the formula(A12) into bromine atom. The conversion into bromine atom may beperformed in the same manner as that of Step 2-5.

Step 2-13

The compounds represented by the formula (A12) can be prepared byreducing the carboxyl group of a marketed compound represented by theformula (A13) into hydroxyl group. As the reducing agent, for example,borane-dimethyl sulfide, borane-tetrahydrofuran and the like can beused, and borane-tetrahydrofuran is preferred. The amount of thereducing agent to be used is, for example, 1 to 5 mol equivalents,preferably 1 to 2 mol equivalents, with respect to the compoundrepresented by the formula (A13), which serves as the starting material.

As the solvent used for the reaction, tetrahydrofuran, diethyl ether andthe like can be used, and preferred examples include tetrahydrofuran. Asfor the reaction temperature, the reaction can be performed usually at 0to 60° C., preferably at 10 to 40° C. Although the reaction time is notparticularly limited, it is, for example, usually 4 to 24 hours,preferably 10 to 18 hours.

Examples of the marketed compound represented by the formula (A13)include, for example, 3-chlorothiophene-2-carboxylic acid,3-bromothiophene-2-carboxylic and the like, and they can be purchasedfrom, for example, Sigma-Aldrich.

Step 3-1

The compounds represented by the formula (15) can be prepared byallowing a vinylation regent to act on a compound represented by theformula (W1) in an organic solvent. As the vinylation reagent, forexample, vinylmagnesium bromide, vinylmagnesium chloride, vinyllithiumand the like can be used, and vinylmagnesium bromide and vinylmagnesiumchloride are preferred. Vinylmagnesium bromide and vinylmagnesiumchloride can be used as a solution in tetrahydrofuran, diethyl ether, ortoluene, and a tetrahydrofuran solution is preferred. As for the amountof the vinylation reagent to be used, the reagent may used in an amountof from equivalent amount to an excess amount, for example, 1 to 10equivalents, preferably 1 to 5 equivalents, with respect to the compoundrepresented by the formula (W1), which serves as the starting material.Examples of the solvent used for the reaction include toluene,tetrahydrofuran, 1,4-dioxane, diethyl ether, 1,2-dimethoxyethane, andmixed solvents of these, and preferred examples include tetrahydrofuranand 1,2-dimethoxyethane. As for the reaction temperature, the reactioncan be performed usually at −78 to 0° C., preferably at −50 to 0° C.Although the reaction time is not particularly limited, it is forexample, usually 0.5 to 24 hours, preferably 1 to 12 hours.

Step 3-2

The compounds represented by the formula (W1) can be prepared from acompound represented by the formula (W2) [in the formula (W2), “hal²”has the same meaning as that defined above] and a compound representedby the formula (12) in the same manner as that of Step 1-3. In thisreaction, the compound represented by the formula (12) can be used in anamount of ⅕ to 20 equivalents, preferably ½ to 10 equivalents, morepreferably 1 to 5 equivalents, with respect to the compound representedby the formula (W2).

Step 3-3

The compounds represented by the formula (W2) can be prepared byreacting a compound represented by the formula (W3) [in the formula(W3), “hal²” has the same meaning as that defined above] withN,O-dimethylhydroxylamine hydrochloride in an organic solvent in thepresence of a base and a dehydration condensation agent. The amount ofN,O-dimethylhydroxylamine hydrochloride used in the reaction of thecompound represented by the formula (W3) and N,O-dimethylhydroxylaminehydrochloride may be equivalent amount to excess amount, for example, 1to 10 equivalents, preferably 1 to 5 equivalents, with respect to thecompound represented by the formula (W3), but the amount can beappropriately determined in consideration of purity, yield, purificationefficiency and the like of the compound represented by the formula (W3).

As the base, for example, triethylamine, diisopropylethylamine,1,4-diazabicyclo[2,2,2]octane, N,N-dimethyl-4-aminopyridine and the likecan be used, and diisopropylethylamine is preferred. The amount of thebase to be used may be equivalent amount to excess amount, for example,1 to 10 equivalents, preferably 1 to 5 equivalents, with respect to thesum of the equivalent of the compound represented by the formula (W3),which serves as the starting material, and the equivalent ofN,O-dimethylhydroxylamine hydrochloride.

As the dehydration condensation agent, there can be usedN,N′-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,N,N′-diisopropylcarbodiimide,N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide p-toluenesulfonate,N,N′-carbonyldiimidazole,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride,1H-benzotriazol-1-yloxy-tris(dimethylphosphonium) hexafluorophosphate,1H-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate,O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate and the like, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is preferred. The amountof the dehydration condensation agent to be used may be equivalentamount to excess amount, for example, 1 to 10 equivalents, preferably 1to 5 equivalents, with respect to the equivalent of the compoundrepresented by the formula (W3), which serves as the starting material.

As an activator, N,N-dimethyl-4-aminopyridine or the like can be added.The amount of the activator to be used may be a catalytic amount toexcess amount, for example, 0.01 to 5 equivalents, preferably 0.1 to 1equivalent, with respect to the equivalent of the compound representedby the formula (W3), which serves as the starting material.

Examples of the solvent used for the reaction include, for example,N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene,toluene, 1,4-dioxane, dichloromethane, chloroform, 1,2-dichloroethane,tetrahydrofuran and the like, and preferred examples includedichloromethane. Two or more kinds of these solvents can also be used asa mixture. As for the reaction temperature, the reaction can beperformed usually at 0 to 100° C., preferably at 20 to 60° C. Althoughthe reaction time is not particularly limited, it is, for example,usually 0.5 to 48 hours, preferably 1 to 24 hours.

Step 3-4

The compounds represented by the formula (14) can be prepared from acompound represented by the formula (W2) [in the formula (W2), “hal²”has the same meaning as that defined above] in the same manner as thatof the method of the step 3-1.

Step 3-5

The compounds represented by the formula (W3) can be prepared by heatinga compound represented by the formula (W4) [in the formula (W4), “hal²”has the same meaning as that defined above] in diluted sulfuric acid. Asthe diluted sulfuric acid used for the reaction, appropriately dilutedconcentrated sulfuric acid or diluted sulfuric acid can be used, and theconcentration thereof is, for example, 0.1 to 15 mol/1, preferably 1 to10 mol/1. The amount of the diluted sulfuric acid to be used can beexcess amount for the compound represented by the formula (W4), and itmay be determined in consideration of yield, purification efficiency andthe like. As for the reaction temperature, the reaction can be performedusually at 20 to 100° C., preferably at 60 to 100° C. Although thereaction time is not particularly limited, it is, for example, usually0.5 to 48 hours, preferably 1 to 24 hours.

Among the compounds represented by the formula (W3), 3-bromophenylaceticacid, 3-iodophenylacetic acid, and 3-bromo-4-fluorophenylacetic acid aremarketed compounds, and can be obtained from Tokyo Chemical Industry.2-(4-Bromothiophen-2-yl)acetic acid is a marketed compound, and can beobtained from APOLLO.

Step 3-6

The compounds represented by the formula (W4) can be prepared byallowing a cyanide to act on a compound represented by the formula (W5)[in the formula (W5), “hal²” has the same meaning as that definedabove]. As the cyanide, sodium cyanide, potassium cyanide, coppercyanide and the like can be used, and sodium cyanide and potassiumcyanide are preferred. The amount of the cyanide to be used may beequivalent amount to excess amount, for example, 1 to 10 equivalents,preferably 1 to 5 equivalents, with respect to the compound representedby the formula (W5), which serves as the starting material. Examples ofthe solvent used for the reaction include, for example, methanol,ethanol, isopropanol, water, mixed solvents of these and the like, andpreferred examples include a mixed solvent of ethanol and water at aratio of 2:1. As for the reaction temperature, the reaction can beperformed usually at 0 to 100° C., preferably at 20 to 100° C. Althoughthe reaction time is not particularly limited, it is, for example,usually 0.5 to 24 hours, preferably 1 to 12 hours.

Step 3-7

The compounds represented by the formula (W5) can be prepared bybrominating a compound represented by the formula (W6) [in the formula(W6), “hal²” has the same meaning as that defined above]. Examples ofthe brominating agent include N-bromosuccinimide, and1,3-dibromo-5,5-dimethylhydantoin, and N-bromosuccinimide is preferred.The amount of the brominating agent to be used may be equivalent amountto excess amount, for example, 1 to 10 equivalents, preferably 1 to 5equivalents, with respect to the compound represented by the formula(W6), which serves as the starting material. Examples of an activator tobe added together with the brominating agent include benzoyl peroxide,tert-butylhydroperoxide, and azobisisobutyronitrile, and benzoylperoxide is preferred. The amount of the activator to be used may be acatalytic amount to excess amount, for example, 0.01 to 2 equivalents,preferably 0.05 to 1 equivalent, with respect to the compoundrepresented by the formula (W6), which serves as the starting material.Examples of the solvent used for the reaction include, for example,carbon tetrachloride, chloroform, 1,2-dichloroethane, mixed solvents ofthese and the like, and preferred examples include carbon tetrachloride.As for the reaction temperature, the reaction can be performed usuallyat 20 to 90° C., preferably at 60 to 90° C. Although the reaction timeis not particularly limited, it is, for example, usually 0.5 to 24hours, preferably 1 to 12 hours.

Examples of the compound represented by the formula (W6) include2-bromo-1,4-dimethylbenzene, 1-bromo-3,5-dimethylbenzene and the like,and these can be purchased as marketed compounds from, for example,Tokyo Chemical Industry.

Step 3-8

The compounds represented by the formula (12) can be prepared byallowing an α-diazophosphonate compound to act on a compound representedby the formula (W7) together with an inorganic base. Examples of thecombination of the α-diazophosphonate compound and the inorganic baseinclude, for example, combinations of dimethyl(diazomethyl)phosphonateand potassium tert-butoxide, dimethyl(diazomethyl)phosphonate and sodiumtert-butoxide, dimethyl(1-diazo-2-oxopropyl)phosphonate and potassiumcarbonate, and dimethyl(1-diazo-2-oxopropyl)phosphonate and sodiumcarbonate, and combination of dimethyl(1-diazo-2-oxopropyl)phosphonateand potassium carbonate is preferred. The amount of theα-diazophosphonate to be used may be equivalent amount to excess amount,for example, 1 to 10 equivalents, preferably 1 to 5 equivalents, withrespect to the compound represented by the formula (W7), which serves asthe starting material. The amount of the inorganic base to be used maybe equivalent amount to excess amount, for example, 1 to 5 equivalents,preferably 1 to 3 equivalents, with respect to the α-diazophosphonate tobe used. Examples of the solvent used for the reaction include, forexample, methanol, ethanol, isopropanol, tert-butanol, mixed solvents ofthese and the like, and preferred examples include methanol. As for thereaction temperature, the reaction can be performed usually at −20 to80° C., preferably at 0 to 60° C. Although the reaction time is notparticularly limited, it is, for example, usually 0.5 to 24 hours,preferably 1 to 12 hours.

Among the compounds represented by the formula (12), 3-ethynylthiopheneand 2-ethynylthiophene are marketed compounds, and can be obtained fromTokyo Chemical Industry.

Step 3-9

4-Phenylthiophene-3-carboaldehyde as the compound represented by theformula (W7) can be prepared by reacting 4-formylthiophene-3-boronicacid and bromobenzene in a solvent in the presence of a base and apalladium catalyst.

As the palladium catalyst, for example, marketed catalysts such astetrakis(triphenylphosphine)palladium,tetrakis(methyldiphenylphosphine)palladium,dichlorobis(triphenylphosphine)palladium,dichlorobis(tri-o-tolylphosphine)palladium,dichlorobis(tricyclohexylphosphine)palladium,dichlorobis(triethylphosphine)-palladium, palladium acetate, palladiumchloride, bis(acetonitrile)palladium chloride,bis(dibenzylideneacetone)palladium,tris(dibenzylideneacetone)dipalladium, andbis(diphenylphosphinoferrocene)palladium chloride may be added to thereaction system as they are, or a catalyst separately prepared frompalladium acetate, tris(dibenzylideneacetone)dipalladium or the like,and an arbitrary ligand, and isolated may be added. A catalystconsidered to actually participate in the reaction may be prepared inthe reaction system by mixing palladium acetate,tris(dibenzylidene-acetone)dipalladium or the like, and an arbitraryligand. The valence of palladium may be 0 or +2. In particular,tris(dibenzylideneacetone)dipalladium(0), palladium(II) acetate and thelike can be mentioned as preferred examples.

As the ligand used for preparing a palladium catalyst from an arbitraryligand, there are exemplified phosphine ligands such astriphenylphosphine, tri(o-tolyl)phosphine, tri(cyclohexyl)phosphine,tri(t-butyl)phosphine, dicyclohexyl-phenylphosphine,1,1′-bis(di-t-butylphosphino)ferrocene,2-dicyclohexylphosphino-2′-dimethylamino-1,1′-biphenyl,2-(di-t-butylphosphino)biphenyl, 2-(dicyclohexyl-phosphino)biphenyl,2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, xantphos,tri(tert-butyl)phosphine,2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl,2-dicyclohexyl-2′,4′,6′-triisopropylbiphenyl, and1,2,3,4,5-pentamethyl-1′-(di-t-butylphosphino)-ferrocene), and preferredexamples include 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl and thelike.

Although the equivalent number of the palladium catalyst to be used maybe equivalent amount or a catalytic amount, it is preferably 0.01 mol %or more, particularly preferably 0.10 to 50.0 mol %, based on thestarting material compound. Examples of the base include, for example,sodium tert-butoxide, cesium carbonate, potassium phosphate and thelike, and potassium phosphate is preferred. The equivalent number of thebase to be used may be equivalent amount or excess amount, for example,1 to 5 equivalents, preferably 1 to 3 equivalents. Examples of thesolvent used for the reaction include, for example, ether type solventssuch as tetrahydrofuran, 1,4-dioxane, and 1,2-dimethoxyethane, toluene,N,N-dimethylformamide, N,N-dimethylacetamide, n-butanol, water, mixedsolvents of these and the like, and a mixed solvent of n-butanol andwater at a ratio of 5:1 can be mentioned as a preferred example. As forthe reaction temperature, the reaction can be performed usually at −20to 120° C., preferably at 0 to 100° C. Although the reaction time is notparticularly limited, it is, for example, usually 0.5 to 48 hours,preferably 1 to 24 hours.

The preparation methods of the compounds of the present invention arenot limited to the methods described herein. For example, the compoundsof the present invention can be prepared by modifying or convertingsubstituents of compounds as precursors of the compounds of the presentinvention using one or a combination of two or more of reactionsdescribed in ordinary chemical articles and the like.

Examples of the preparation method for the compounds of the presentinvention which contain an asymmetric carbon include a preparationmethod based on asymmetric reduction, a method of using a commerciallyavailable starting material (or starting material that can be preparedby a known method or a method similar to a known method) of which moietycorresponding to the asymmetric carbon is originally optically activeand the like. A method is also available in which a compound of thepresent invention or a precursor thereof is separated as an opticallyactive isomer by a conventional method. Examples of such a methodinclude, for example, a method utilizing high performance liquidchromatography (HPLC) using a chiral column, the classical fractionalcrystallization for separation of optically active substances comprisingformation of a salt with an optically active regent, separation byfractional crystallization or the like, and conversion of the salt intoa compound of free form, a method comprising condensation with anoptically active regent to form a diastereomer, followed by separation,purification, and decomposition of the produced diastereomer and thelike. When a precursor is separated to obtain an optically activesubstance, an optically active compound of the present invention canthen be prepared by performing the aforementioned preparation methods.

When a compound of the present invention contains an acidic functionalgroup such as carboxyl group, phenolic hydroxyl group, or tetrazolering, the compound can be converted into a pharmaceutically acceptablesalt (e.g., inorganic salts with sodium and the like, or organic saltswith triethylamine and the like) by a known means. For example, when aninorganic salt is to be obtained, it is preferable to dissolve thecompound of the present invention in water containing at least 1equivalent of hydroxide, carbonate, bicarbonate or the likecorresponding to the desired inorganic salt. For the reaction, awater-miscible inactive organic solvent such as methanol, ethanol,acetone, and dioxane may be mixed. For example, by using sodiumhydroxide, sodium carbonate, or sodium hydrogencarbonate, a solution ofsodium salt can be obtained.

When a compound of the present invention contains amino group, anotherbasic functional group, or an aromatic ring which itself has a basicproperty (e.g., pyridine ring and the like), the compound can also beconverted into a pharmaceutically acceptable salt (e.g., salt with aninorganic acid such as hydrochloric acid, or salt with an organic acidsuch as acetic acid) by a known means. For example, when a salt with aninorganic acid is to be obtained, it is preferable to dissolve thecompound of the present invention in water containing at least 1equivalent of a desired inorganic acid. For the reaction, awater-miscible inactive organic solvent such as methanol, ethanol,acetone, and dioxane may be mixed. For example, by using hydrochloricacid, a solution of hydrochloride can be obtained.

If a solid salt is desired, the solution may be evaporated, or awater-miscible organic solvent having polarity to some extent, such asn-butanol or ethyl methyl ketone, can be added to the solution to obtaina solid salt.

The various compounds disclosed by the present invention can be purifiedby known methods such as variety of chromatography techniques (columnchromatography, flash column chromatography, thin layer chromatography,high performance liquid chromatography).

The compounds of the present invention according to a certain embodimenthave an EP₄ agonist activity, and can be used as EP₄ agonist. That is,the compounds of the present invention according to a certain embodimentcan be used as a medicament for prophylactic and/or therapeutictreatment of a disease relating to EP₄ receptor agonization. The diseaserelating to EP₄ receptor agonization is, more precisely, a disease forwhich EP₄ receptor agonization is effective, and more specifically, itis not particularly limited so long as it is a disease that can beprevented and/or treated by raising the cAMP production amount inosteoblasts.

The EP₄ agonist activity can be measured by, for example, the methodsdescribed below. That is, a method of confirming promotion of the cAMPproduction in a human EP₄ receptor-expressing cell can be mentioned. Asanother embodiment, a method of confirming osteogenesis-promoting actionbased on promotion of cAMP production in the presence of cyclooxygenase2 (COX-2) inhibitor in rat marrow cells can be mentioned. As stillanother embodiment, a method of confirming activity of binding to thehuman EP₄ receptor can be mentioned. As the method for confirmingosteogenesis-promoting action based on promotion of the cAMP production,specifically, the method described in Test Examples 1 mentioned latercan be exemplified.

The EP₄ agonist activity that can be confirmed by the method describedin Test Example 1 is, for example, 10 nM or lower, preferably 1 nM orlower, more preferably 0.6 nM or lower, still more preferably 0.3 nM orlower, particularly preferably 0.1 nM or lower, most preferably 0.05 nMor lower.

The compounds of the present invention according to a certain embodimentshow high specificity (selectivity) for EP₄. The selectivity for EP₄ canbe evaluated by, for example, performing measurement of agonist activityand receptor binding test using cells that express each of human EP₁,EP₂, and EP₃ receptors to calculate ratio of the IC₅₀ values(concentration of the compound of the present invention at which thebinding of [³H]PGE₂ and the receptor is suppressed by 50%), or the Kivalues. Specifically, the method described in Test Example 2 can beexemplified.

Ratio of IC ₅₀ value(time)=IC ₅₀ for each receptor/IC ₅₀ for EP ₄

Ratio of Ki value(time)=Dissociation constant Ki for eachreceptor/Dissociation constant Ki for EP ₄

In order to avoid side reactions, it is preferred that the compounds ofthe present invention according to a certain embodiment show highspecificity for EP₄. For example, the ratio of the IC₅₀ value or Kivalue should be 10 times or larger, preferably 100 times or larger, morepreferably 1,000 times or larger, further preferably 3000 times orlarger, particularly preferably 10,000 times or larger.

It is also preferred that the compounds of the present inventionaccording to a certain embodiment selectively act on or bind to the EP₄receptor, but do not act on or bind to the EP₁ receptor, EP₂ receptor,and EP₃ receptor, as well as DP receptor, FP receptor, IP receptor, TPreceptor, PPARα receptor, PPARδ receptor, PPARγ receptor, S1P receptors(e.g., S1P1 receptor, S1P2 receptor, S1P3 receptor and the like), LTB4receptors (e.g., BLT1, BLT2 and the like), LPA receptors (e.g., LPA1receptor, LPA2 receptor, LPA3 receptor and the like), and cannabinoidreceptors (e.g., CB1 receptor, CB2 receptor and the like), or act on orbind to these more weakly compared with the action on or binding to theEP₄ receptor.

The disease relating to the EP₄ receptor agonization is not particularlylimited so long as it is a disease for which agonization of the EP₄receptor is effective, and specific examples include, for example, bonefracture and bone defect.

The compounds of the present invention according to a certain embodimenthave an osteogenesis-promoting action as shown in the test examplesmentioned later, and are useful as an active ingredient of a medicament.The compounds of the present invention according to a certain embodimentare used for, in particular, therapeutic treatment and/or promotion ofhealing of fracture or bone defect, and preferably used for therapeutictreatment and/or promotion of healing of fracture. As anotherembodiment, they are also preferably used for therapeutic treatmentand/or promotion of healing of bone defect.

Usefulness of the medicament of the present invention according to acertain embodiment for therapeutic treatment and/or promotion of healingof fracture or bone defect can be confirmed by using a closed fracturemodel or a partial or most long bone defect model. Specifically, themethod described in Test Example 5 is exemplified.

The medicament of the present invention according to a certainembodiment can be expected to exhibit a systemic bone density-increasingaction and bone strength-increasing action, or local boneregeneration/osteoanagenesis-promoting action. Theosteogenesis-promoting action of the compounds of the present inventionaccording to a certain embodiment can be evaluated by, for example,using bone marrow cells isolated from experimental animals such as rats,or human, and cultured, and using number of formed calcified bone-likenodules, alkaline phosphatase activity, which is a differentiationmarker of osteoblasts or the like as a marker. It can also be evaluatedby using pathological model animals such as reduced bone mass model ratssubjected to sciatic nerve resection and ovariectomy or the like, andbone density or bone strength of the appendicular skeletons or the likeas a marker. It can also be evaluated by using a rat long bone closedfracture model or bone cut model prepared by invasive operation, a modelin which bone defect is created in an arbitrary region or the like, andosteogenesis, bone union rate, bone strength of restored bone or thelike as a marker.

Fracture means a condition that a bone is partially or completelyinterrupted or deformed caused by an external force. The bone that maysuffer from fracture is not particularly limited, so long as it is of apatient whose osseous tissue is damaged, and examples include, forexample, facial bones (orbital bone, cheek bone, mandible), trunk bones(rib, pelvis, cervical vertebra, thoracic vertebra, lumbar vertebra,sacral bone, coccygeal bone), bones of the upper limb (scapula,clavicle, humerus, elbow, radius, ulna, scaphoid, hamatum, metacarpus,phalanx), bones of the lower limb (hip joint, femur, tibia, fibula,ankle joint, calcaneus, scaphoid, metatarsus) and the like, and theobjective bone may be of any part. The type of the damage of osseoustissue is not particularly limited, and promotion of union of bones infracture (complete fracture, incomplete fracture, simple fracture,comminuted fracture and the like), or bone intentionally cut inosteotomy or bone extension surgery adapted as one of the surgicaltreatment means is also included. Femoral neck fracture, vertebralcompression fracture, fracture of the distal radius, fracture of humerusproximal end and the like, of which causative disease is osteoporosis,are also included in the scope of the fracture mentioned above.

Bone defect means various bone diseases themselves such as osteoncus,osteomyelitis, traumatic injury, chronic articular disease, prolongedhealing after fracture, and slack of artificial joint, or a conditionthat a defect of a bone is formed by surgically excising a lesion in atreatment of such diseases. The part thereof is not particularlylimited, so long as it is of a patient who has been obliged to have abone defect. Examples include facial bones (orbital bone, cheek bone,mandible), trunk bones (rib, pelvis, cervical vertebra, thoracicvertebra, lumbar vertebra, sacral bone, coccygeal bone), bones of theupper limb (scapula, clavicle, humerus, elbow, radius, ulna, scaphoid,hamatum, metacarpus, phalanx), bones of the lower limb (hip joint,femur, tibia, fibula, ankle joint, calcaneus, scaphoid, metatarsus) andthe like, and the objective bone may be of any part. The type of bonedefect is not particularly limited, and bone defects of any type such asa condition that intermediate part of a bone is extensively defective,and a condition that a bone becomes partially defective because ofcomminuted fracture are included.

The medicament of the present invention according to a certainembodiment can be used as a bone union-promoting agent at the time ofsurgical therapeutic interventions. For example, it can be used in spine(cervical vertebra, thoracic vertebra, and lumbar vertebra) fixation,denatured scoliosis surgery, joint replacement, vertebral canalexpansion, osteotomy, bone extension surgery, cranial bone defectcompensation, cranioplasty, ilium spacer fixation with bony support,heterologous bone grafting, homologous bone grafting, autologous bonegrafting, and bone graft substitute therapy, as well as bone restorationand/or bone reconstruction after surgical extraction of primarymalignant tumor or bone metastasis lesion, which are exemplified asmedical interventions.

The medicament of the present invention according to a certainembodiment is preferably used as an osteogenesis-promoting agent. Themedicament of the present invention according to a certain embodiment ismore preferably used for therapeutic treatment and/or promotion ofhealing of fracture or bone defect. Further, the medicament of thepresent invention according to a certain embodiment is extremelypreferably used for prophylactic and/or therapeutic treatment offracture. In addition, it can be easily understood by those skilled inthe art that a medicament for preventing or suppressing progress of apathological condition falls within the scope of the medicament forprophylactic and/or therapeutic treatment referred to in the presentinvention, as the case may be.

The medicament of the present invention according to a certainembodiment can be prepared as a medicament containing a compoundrepresented by the formula (1) or a pharmaceutically acceptable saltthereof as an active ingredient, and for example, a medicamentcontaining a compound or a pharmaceutically acceptable salt thereof thatis metabolized in a living body to produce the compound represented bythe formula (1) or a pharmaceutically acceptable salt thereof when it isadministered as a prodrug also falls within the scope of the medicamentof the present invention.

Although administration route of the medicament of the present inventionaccording to a certain embodiment is not particularly limited, theadministration scheme can be appropriately selected from, for example,oral administration, subcutaneous administration, intracutaneousadministration, intramuscular injection, intravenous administration,pernasal administration, intravaginal administration, intrarectaladministration, local administration to an affected part and the like.The local administration to an affected part is one of the preferredadministration schemes.

As the medicament of the present invention, a compound represented bythe formula (1) or a pharmaceutically acceptable salt thereof, per se,may be used. However, it is preferable to add one or more kinds ofpharmaceutically acceptable carriers to the compound represented by theformula (1) or a pharmaceutically acceptable salt thereof to prepare apharmaceutical composition and administer the composition. Further, asthe active ingredient of the medicament of the present invention, ahydrate or solvate of a compound represented by the general formula (I)or a pharmaceutically acceptable salt thereof may be used.

Examples of dosage form used for preparing the aforementionedpharmaceutical composition include tablet, powder, granule, syrup,suspension, capsule, inhalant, injection and the like. For themanufacture of them, various carriers suitable for these preparationsare used. For example, examples of the carrier for oral preparationsinclude excipients, binders, lubricants, fluid accelerators, andcolorants. Examples of the method for using the composition as aninhalants include a method of inhaling powder of the pharmaceuticalcomposition or a liquid dosage form prepared by dissolving or suspendingthe pharmaceutical composition in a solvent as it is, a method ofinhaling mist thereof by using a sprayer called atomizer or nebulizerand the like. When the composition is formulated as an injection,distilled water for injection, physiological saline, glucose aqueoussolution, vegetable oil for injection, propylene glycol, polyethyleneglycol and the like can generally be used as a diluent. Disinfectants,antiseptics, stabilizers, isotonic agents, soothing agents and the likemay be further added, as required. A clathrate compound in which thecompound of the present invention is clathrated in cyclodextrin may beprepared, and used as the medicament of the present invention.

When the medicament of the present invention according to a certainembodiment is administered, an appropriate dosage form can be suitablychosen and administered via an appropriate route. For example, it can beorally administered in the form of tablet, powder, granule, syrup,suspension, capsule or the like. The medicament can also be administeredvia the respiratory tract in the form of an inhalant. In addition, themedicament can be subcutaneously, intracutaneously, intravascularly,intramuscularly, or intraperitoneally administered in the form of aninjection including drip infusion. Furthermore, the medicament can betransmucosally administered in the form of sublingual tablet,suppository or the like, and can be percutaneously administered in theform of gel, lotion, ointment, cream, spray or the like. In addition,the medicament can also be administered as a prolonged action drug, forexample, a sustained-release injection, or an embedding formulation(e.g., film formulation and the like).

When the medicament of the present invention according to a certainembodiment is locally administered, the medicament can be directlyadministered to a local site such as fracture part. In such a case, thecompound can be directly injected to the local site together with anappropriate non-hydrophilic solvent, or the compound can also beformulated in an appropriate carrier such as biodegradable polymers, andused as a medicament molded into a rod shape, needle shape, sphericalshape, film shape or the like, or in the form of ointment, cream, orgel, or sustained release preparation by embedding or injecting themedicament in a local site such as fracture part. Examples of thebiodegradable high molecular polymer include, for example, aliphaticacid polyesters (polymers and copolymers of one or more kinds ofα-hydroxycarboxylic acids, hydroxydicarboxylic acids, lacticacid/caprolactone, valerolactone and the like, and mixtures thereof),derivatives thereof (polylactic acids, polyglycolic acids, blockpolymers of polyethylene glycol and the like), poly-α-cyanoacrylic acidesters, poly-β-hydroxybutyric acids, polyalkylene oxalates,polyortho-esters, polyortho-carbonates, polyocarbonates, polyaminoacids, hyaluronic acid esters, polystyrene groups, polymethacrylicacids, copolymers of acrylic acid and methacrylic acid, polyamino acids,decyne stearate, ethylcellulose, acetylcellulose, nitrocellulose, maleicanhydride copolymers, ethylene vinyl acetate copolymers, polyvinylacetates, polyacrylamides, collagen, gelatin, fibrin, bone meal, bonecement and the like.

The biodegradable high molecular polymer may consist of one kind ofsubstance, or a copolymer, a complex, or a simple mixture of two or morekinds of substances, and the polymerization scheme thereof may be any ofrandom, block, and graft polymerizations.

The medicament of the present invention according to a certainembodiment can also be applied or adsorbed on an artificial bone(implant) consisting of a highly biocompatible material (metal, calcium,ceramics, polymer materials and the like), bone prosthesis(hydroxyapatite, β-tricalcium phosphate and the like) or the liketogether with an appropriate solvent or carrier, or embedded therein,and administered to a local site.

The administration period of the medicament of the present inventionaccording to a certain embodiment is not particularly limited. Inprinciple, the medicament is administered during a period where it isjudged that clinical symptoms of a disease are expressed, and it iscommon to continue the administration for several weeks to one year.However, it is also possible to extend the administration perioddepending on pathological conditions, or continue the administrationeven after recovery from the clinical symptoms. The medicament may alsobe prophylactically administered by a decision of a clinician even ifany clinical symptom is not expressed. The dose of the medicament of thepresent invention according to a certain embodiment is not particularlylimited. When the medicament of the present invention is directlyadministered to a local site such as fracture part, 0.01 to 1,000 μg ofthe active ingredient can be administered to an adult per eachadministration. As for administration frequency in the above case, themedicament may be administered at a frequency of every 6 months to everyday, and the medicament may preferably be administered once per 3 monthsto once per month, or once per week.

The daily dose and/or dose per one time, administration period, andadministration frequency may be suitably increased or decreaseddepending on various conditions such as age, weight, degree of physicalhealthiness of a patient, type and severity of a disease to be treated,administration route, and dosage form (sustained release property ofcarrier for active ingredient and the like).

When the medicament of the present invention according to a certainembodiment is used for therapeutic treatment and/or promotion of healingof fracture or bone defect, or prophylactic and/or therapeutic treatmentof fracture, the medicament of the present invention according to acertain embodiment can be used together with one or more kinds ofmedicaments selected from the group consisting of bone-activatingagents, osteogenesis-promoting agents, bone resorption-suppressingagents, bone metabolism-improving agents, sexual hormone preparations,and calcium preparations, simultaneously or at different times. Further,the medicament of the present invention according to a certainembodiment can also be prepared as a so-called combined drug togetherwith the medicaments exemplified above, and then administered. Theaforementioned combined drug may be in a dosage form as a completemixture of the active ingredients similar to typical compositions ofsuch type, as well as a dosage form, kit, or package including anon-mixed combination of ingredients separately administered from two ormore containers each of which contains each active ingredient.

Examples of the bone-activating agents usable in combination with themedicament of the present invention according to a certain embodimentinclude, for example, vitamin D or vitamin D derivatives such ascalcitriol, alfacalcidol, OCT, 2MD, and ED-71, examples of theosteogenesis-promoting agents include, for example, menatetrenone,teriparatide, somatropin, insulin-like growth factor-I (IGF-I), bonemorphogenetic proteins (BMPs), basic fibroblast growth factor (bFGF),transforming growth factor-β (TGF-β), EP₂ agonist, LRP5 agonist,anti-SOST antibody, GSK-3 inhibitor, Dkk1 inhibitor, calcilytics, growthhormone secretagogues and the like, examples of the boneresorption-suppressing agents include, for example, elcatonin,calcitonin salmon, etidronate, pamidronate, clodronate, alendronate,incadronate, risedronate, minodronate, ibandronate, cathepsin Kinhibitors, osteoprotegerin, anti-RANKL antibodies and the like,examples of the bone metabolism-improving agents include, for example,fluoride, strontium ranelate, ipriflavone and the like, examples of thesexual hormone preparations include, for example, estriol, estradiol,conjugated estrogen, progesterone, medroxyprogesterone, testosterone,metyltestosterone, mestanolone, stanozolol, metenolone, nandrolone,selective estrogen receptor modulators (SERM: raloxifen, lasofoxifene,bazedoxifene, ospemifene, arzoxifene, CHF4227, PSK-3471 and the like),selective androgen receptor modulators (SARM) and the like, and examplesof the calcium preparations include, for example, calcium carbonate,calcium lactate, calcium gluconate, calcium acetate, calcium chloride,calcium citrate, calcium hydrogenphosphate, calcium L-aspartate and thelike. It can also be used together with various kinds of drugs for bonediseases to be created in the future. These combined drugs are notlimited so long as the combinations are clinically meaningful.

The compounds of the present invention according to a certain embodimentinclude compounds showing superior safety (concerning various toxicitiesand safety pharmacology), pharmacokinetic performance and the like, andusefulness thereof as an active ingredient of a medicament can beconfirmed by, for example, the methods shown below.

Examples of tests concerning safety include, for example, those listedbelow. However, they are not limited to these examples. Examples includecytotoxic tests (tests using HL60 cells, hepatocytes and the like),genotoxicity tests (Ames test, mouse lymphoma TK test, chromosomalaberration test, micronucleus test and the like), skin sensitizationtests (Buehler method, GPMT method, APT method, LLNA test and the like),skin photosensitization tests (adjuvant and strip method and the like),eye irritation tests (single instillation, short-term continuationinstillation, repetitive instillation and the like), safety pharmacologytests for the cardiovascular system (telemetry method, APD method, hERGinhibition assay and the like), safety pharmacology tests for thecentral nervous system (FOB method, modified version of Irwin method andthe like), safety pharmacology tests for the respiratory system(measurement method utilizing a respiratory function measuringapparatus, measurement method utilizing a blood gas analyzer and thelike), general toxicity tests, reproductive and developmental toxicitytests and the like.

Examples of tests concerning pharmacokinetic performance include, forexample, those listed below. However, they are not limited to theseexamples. Examples include cytochrome P450 enzyme inhibition orinduction tests, cell permeability tests (tests using CaCO-2 cells, MDCKcells and the like), drug transporter ATPase assay, oral absorptiontests, blood concentration transition measurement tests, metabolismtests (stability test, metabolite molecular species test, reactivitytest and the like), solubility tests (solubility test based on turbiditymethod and the like) and the like.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a cytotoxic test. Examples of thecytotoxic test include methods utilizing various cultured cells, forexample, HL-60 cells, which are human preleukemia cells, primaryisolated cultured cells of hepatocytes, a neutrophil fraction preparedfrom human peripheral blood and the like. Although the test can becarried out by the method described below, the method is not limitedonly to the following description. Cells are prepared as a suspension of10⁵ to 10⁷ cells/ml, and the suspension is added to microtubes ormicroplate in a volume of 0.01 to 1 mL. To the suspension, a solutiondissolving a compound is added in a volume of 1/100 to 1 fold volume ofthe cell suspension, and the cells were cultured in a cell culturemedium having a final concentration of the compound of 0.001 to 1,000 μMfor 30 minutes to several days at 37° C. under 5% CO₂. After terminatingthe culture, survival rate of the cells is evaluated by using the MTTmethod, WST-1 method (Ishiyama, M., et al., In Vitro Toxicology, 8, p.187, 1995) or the like. By measuring cytotoxicity of a compound tocells, usefulness of the compound as an active ingredient of amedicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a genotoxicity test. Examples ofthe genotoxicity test include, the Ames test, mouse lymphoma TK test,chromosomal aberration test, micronucleus test and the like. The Amestest is a method of determining reverse mutation by culturing Salmonellaor Escherichia bacteria of designated species on a culture dish or thelike added with a compound (refer to IYAKUSHIN (Notification by thechief of Evaluation and Licensing Division, Pharmaceutical and MedicalSafety Bureau, Ministry of Health, Labor and Welfare, Japan), No. 1604,1999, “Guideline for Genotoxicity Test”, II-1. Genotoxicity Test and thelike). The mouse lymphoma TK test is a genetic mutation abilitydetection test targeting the thymidine kinase gene of the mouse lymphomaL5178Y cell (refer to IYAKUSHIN No. 1604, 1999, “Guideline forGenotoxicity Test”, 11-3. Mouse Lymphoma TK Test; Clive, D. et al.,Mutat. Res., 31, pp. 17-29, 1975; Cole, J., et al., Mutat. Res., 111,pp. 371-386, 1983 and the like). The chromosomal aberration test is amethod for determining activity of causing chromosomal aberration byculturing mammalian cultured cells in the presence of a compound, thenafter fixation of the cells, staining and observing chromosomes of thecells (refer to IYAKUSHIN No. 1604, 1999, “Guideline for GenotoxicityTest”, 11-2. Chromosomal Aberration Test Utilizing Mammalian CulturedCells and the like). The micronucleus test is a method of evaluatingmicronucleus-forming ability caused by chromosomal aberration, and amethod of using a rodent (in vivo test) (IYAKUSHIN No. 1604, 1999,“Guideline for Genotoxicity Test”, 11-4. Micronucleus Test Using Rodent;Hayashi M. et al., Mutat. Res., 312, pp. 293-304, 1994; Hayashi, M. etal., Environ. Mol. Mutagen., 35, pp. 234-252, 2000), a method of usingcultured cells (in vitro test) (Fenech M., et al., Mutat. Res., 147, pp.29-36, 1985; Miller, B., et al., Mutat. Res., 392, pp. 45-59, 1997) andthe like are available. By elucidating genotoxicity of a compound usingone or more of these methods, usefulness of the compound as an activeingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a skin sensitization test. Skinsensitization tests include, as the skin sensitization tests usingguinea pig, the Buehler method (Buehler, E. V., Arch. Dermatol., 91, pp.171-177, 1965), GPMT method (maximization method, Magnusson B., et al.,J. Invest. Dermatol., 52, pp. 268-276, 1969), APT method (adjuvant andpatching method (Sato, Y. et al., Contact Dermatitis, 7, pp. 225-237,1981)) and the like. Further, as the skin sensitization test usingmouse, the LLNA (local lymph node assay) method (OECD Guideline for thetesting of chemicals 429, Skin sensitization 2002; Takeyoshi, M. et al.,Toxicol. Lett., 119 (3), pp. 203-8, 2001; Takeyoshi, M. et al., J. Appl.Toxicol., 25 (2), pp. 129-34, 2005) and the like are available Byelucidating skin sensitization property of a compound using one or moreof these methods, usefulness of the compound as an active ingredient ofa medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a skin photosensitization test.Examples of the skin photosensitization test include a skinphotosensitization test using guinea pig (refer to “Drug NonclinicalTest Guideline Commentary 2002”, Yakuji Nippo, published on 2002, 1-9:Skin Photosensitization Test and the like) and the like, and examples ofthe method include the adjuvant and strip method (Ichikawa, H. et al.,J. Invest. Dermatol., 76, pp. 498-501, 1981), Harber method (Harber, L.C., Arch. Dermatol., 96, pp. 646-653, 1967), Horio method (Horio, T., J.Invest. Dermatol., 67, pp. 591-593, 1976), Jordan method (Jordan, W. P.,Contact Dermatitis, 8, pp. 109-116, 1982), Kochever method (Kochever, I.E. et al., J. Invest. Dermatol., 73, pp. 144-146, 1979), Maurer method(Maurer, T. et al., Br. J. Dermatol., 63, pp. 593-605, 1980), Morikawamethod (Morikawa, F. et al., “Sunlight and Man”, Tokyo Univ. Press,Tokyo, pp. 529-557, 1974), Vinson method (Vinson, L. J., J. Soc. Cosm.Chem., 17, pp. 123-130, 1966) and the like. By elucidating skinphotosensitization property of a compound using one or more of thesemethods, usefulness of the compound as an active ingredient of amedicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, an eye irritation test. Examplesof the eye irritation test include the single instillation test methodusing rabbit eyes, monkey eyes and the like (instillation of one time),short term continuous instillation test method (instillation of multipletimes in a short period of time with equal intervals), repetitiveinstillation test method (repetitive intermittent instillation overseveral days to several 10 days) and the like, and a method ofevaluating eye irritation symptoms during a certain period of time afterinstillation according to the improved Draize scores (Fukui, N. et al.,Gendai no Rinsho, 4 (7), pp. 277-289, 1970) and the like are availableBy elucidating eye irritation of a compound using one or more of thesemethods, usefulness of the compound as an active ingredient of amedicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a safety pharmacology test for thecardiovascular system. Examples of the safety pharmacology test for thecardiovascular system include the telemetry method (method for measuringinfluence of administration of a compound under no anesthetization onelectrocardiogram, heart rate, blood pressure, blood stream and the like(Electrocardiogram, Echocardiography, Blood Pressure and PathologicalTests of Animals for Fundamental and Clinical Medicine, edited by SuganoS., Tsubone H., Nakada Y, published on 2003, Maruzen), APD method(method for measuring cardiac muscle cell action potential retentiontime (Muraki, K. et al., AM. J. Physiol., 269, H524-532, 1995; Ducic, I.et al., J. Cardiovasc. Pharmacol., 30 (1), pp. 42-54, 1997)), hERGinhibition evaluation method (patch clamping method (Chachin, M. et al.,Nippon Yakurigaku Zasshi, 119, pp. 345-351, 2002), binding assay method(Gilbert, J. D. et al., J. Pharm. Tox. Methods, 50, pp. 187-199, 2004),Rb⁺ efflex assay method (Cheng, C. S. et al., Drug Develop. Indust.Pharm., 28, pp. 177-191, 2002), Membrane potential assay method (Dorn,A. et al., J. Biomol. Screen., 10, pp. 339-347, 2005) and the like. Byelucidating influence on the cardiovascular system of a compound usingon one or more of these methods, usefulness of the compound as an activeingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a safety pharmacology test for thecentral nervous system. Examples of the safety pharmacology test for thecentral nervous system include the FOB method (Functional ObservationalBattery, Mattson, J. L. et al., J. American College of Technology, 15(3), pp. 239-254, 1996)), modified version of Irwin method (method forevaluating observation of general symptoms and behavior (Irwin, S.,Comprehensive Observational Assessment (Berl.) 13, pp. 222-257, 1968))and the like. By elucidating action on the central nervous system of acompound using one or more of these methods, usefulness of the compoundas an active ingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a safety pharmacology test for therespiratory system. Examples of the safety pharmacology test for therespiratory system include the measurement method using a respiratoryfunction measuring apparatus (method of measuring respiration rate,single ventilation volume, minute ventilation and the like, Drorbaugh,J. E. et al., Pediatrics, 16, pp. 81-87, 1955; Epstein, M. A. et al.,Respir. Physiol., 32, pp. 105-120, 1978), measurement method of using ablood gas analyzer (method of measuring blood gas, hemoglobin oxygensaturation and the like, Matsuo, S., Medicina, 40, pp. 188-, 2003) andthe like. By elucidating action on the respiratory system of a compoundusing one or more of these methods, usefulness of the compound as anactive ingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a general toxicity test. Thegeneral toxicity test is a method of orally or intravenouslyadministering a compound dissolved or suspended in an appropriatesolvent once or repetitively (over several days) to a rodent such as ratand mouse or non-rodent such as monkey and dog, and evaluatingobservation of general conditions, clinicochemical changes,pathohistological changes and the like of the administered animal. Byelucidating general toxicity of a compound using these methods,usefulness of the compound as an active ingredient of a medicament canbe confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a reproductive and developmentaltoxicity test. The reproductive and developmental toxicity test is atest for examining induction of harmful effect caused by a compound onthe reproductive and developmental processes by using a rodent such asrat and mouse, or non-rodent such as monkey and dog (refer to “DrugNonclinical Test Guideline Commentary 2002”, Yakuji Nippo, published on2002, 1-6: Reproductive and Developmental Toxicity Test and the like).Examples of the reproductive and developmental toxicity test includetests concerning fertility and early embryogenesis up to nidation, testsconcerning development and maternal functions before and after birth,tests concerning embryogenesis and fetal development (refer to IYAKUSHINNo. 1834, 2000, Appendix, “Guideline for Drug Toxicity Test”, [3]Reproductive and Developmental Toxicity Test and the like) and the like.By elucidating reproductive and developmental toxicity of a compoundusing these methods, usefulness of the compound as an active ingredientof medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a cytochrome P450 enzymeinhibition or induction test (Gomez-Lechon, M. J. et al., Curr. DrugMetab., 5 (5), pp. 443-462, 2004). Examples of the cytochrome P450enzyme inhibition or induction test include, for example, the method ofdetermining in vitro whether a compound inhibits activity of acytochrome P450 enzyme by using a cytochrome P450 enzyme of eachmolecular species purified from cells or prepared by using a geneticrecombinant, or a human P450 expression system microsome (Miller, V. P.et al., Ann. N.Y. Acad. Sci., 919, pp. 26-32, 2000), method of measuringchanges of expression of cytochrome P450 enzyme of each molecularspecies or enzyme activity by using human liver microsomes or disruptedcell suspension (Hengstler, J. G. et al., Drug Metab. Rev., 32, pp.81-118, 2000), method of extracting RNA from human hepatocytes exposedto a compound, and comparing mRNA expression amount with that of acontrol to investigate enzyme induction ability of the compound (Kato,M. et al., Drug Metab. Pharmacokinet., 20 (4), pp. 236-243, 2005) andthe like. By elucidating action of a compound on inhibition or inductionof cytochrome P450 enzyme using one or more of these methods, usefulnessof the compound as an active ingredient of a medicament can beconfirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a cell permeability test. Examplesof the cell permeability test include, for example, the method ofmeasuring cell membrane permeability of a compound in an in vitro cellculture system using CaCO-2 cells (Delie, F. et al., Crit. Rev. Ther.Drug Carrier Syst., 14, pp. 221-286, 1997; Yamashita, S. et al., Eur. J.Pham. Sci., 10, pp. 195-204, 2000; Ingels, F. M. et al., J. Pham. Sci.,92, pp. 1545-1558, 2003), method of measuring cell membrane permeabilityof a compound in an in vitro cell culture system using MDCK cells(Irvine, J. D. et al., J. Pham. Sci., 88, pp. 28-33, 1999) and the like.By elucidating cell permeability of a compound using one or more ofthese methods, usefulness of the compound as an active ingredient of amedicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a drug transporter ATPase assayfor ATP-binding cassette (ABC) transporter. Examples of the drugtransporter ATPase assay include the method of examining whether acompound is a substrate of P-glycoprotein (P-gp) by using a P-gpbaculovirus expression system (Germann, U. A., Methods Enzymol., 292,pp. 427-41, 1998) and the like Furthermore, the usefulness can also beverified by performing, for example, a transport test using oocytescollected from African clawed frog (Xenopus laevis) as a solute carrier(SLC) transporter. Transport tests include a method of examining whethera test compound is a substrate of OATP2 using OATP2-expressing oocytes(Tamai I. et al., Pharm. Res., 2001 September; 18 (9), 1262-1269) andthe like. By elucidating action of a compound on the ABC transporter orSLC transporter using these methods, usefulness of the compound as anactive ingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, an oral absorption test. Examplesof the oral absorption test include a method of orally administering acompound of a certain amount dissolved or suspended in an appropriatesolvent to a rodent, monkey, dog or the like, and measuring blood levelof the compound after the oral administration over time to evaluateblood transition of the compound by oral administration using theLC-MS/MS method (“Newest Mass Spectrometry for Life Science”, KodanshaScientific, 2002, edited by Harada K. et al and the like) and the like.By elucidating oral absorption of a compound using these methods,usefulness of the compound as an active ingredient of a medicament canbe confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a blood concentration transitionmeasurement test. Examples of the blood concentration transitionmeasurement test include a method of orally or parenterally (e.g.,intravenously, intramuscularly, intraperitoneally, subcutaneously,transdermally, by instillation, transnasally and the like) administeringa compound to a rodent, monkey, dog or the like, and measuring change ofthe blood level of the compound over time after the administration usingthe LC-MS/MS method (“Newest Mass Spectrometry for Life Science”,Kodansha Scientific, 2002, edited by Harada K. et al and the like) andthe like. By elucidating blood concentration transition of a compoundusing these methods, usefulness of the compound as an active ingredientof a medicament can be confirmed. In the case of, in particular, localadministration among the parenteral administrations, in order to avoidside reactions, the compounds of the present invention according to acertain embodiment showing low blood concentration after administrationthereof may be preferred.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a metabolic test. Examples of themetabolic test include the blood stability test method (method ofpredicting metabolic clearance in vivo on the basis of metabolic rate ofa compound in hepatic microsomes of human or other animal species (referto Shou, W. Z. et al., J. Mass Spectrom., 40 (10) pp. 1347-1356, 2005;Li, C. et al., Drug Metab. Dispos., 34 (6), 901-905, 2006 and the like),metabolite molecular species test method, reactive metabolite testmethod and the like. By elucidating metabolic profile of a compound byusing one or more of these methods, usefulness of the compound as anactive ingredient of a medicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by performing, for example, a solubility test. As the methodfor evaluating solubility in water, the methods of confirming thesolubility under acidic conditions, neutral conditions, or basicconditions are exemplified, and confirming change of solubilitydepending on the presence or absence of bile acid is also included.Examples of the solubility test include the solubility test based on theturbidity method (Lipinski, C. A. et al., Adv. Drug Deliv. Rev., 23, pp.3-26, 1997; Bevan, C. D. et al., Anal. Chem., 72, pp. 1781-1787, 2000)and the like. By elucidating solubility of a compound using thesemethods, usefulness of the compound as an active ingredient of amedicament can be confirmed.

Usefulness of the compounds of the present invention according to acertain embodiment as an active ingredient of a medicament can beconfirmed by examining, for example, upper gastrointestinal injury,renal dysfunction and the like. As a pharmacological test for the uppergastrointestinal tract, actions on gastric mucosa can be investigated byusing a starved rat gastric mucosa injury model. Examples ofpharmacological test for kidney functions include renal blood flow andglomerular filtration rate measuring method [Physiology, 18th edition,Bunkodo, 1986, Chapter 17] and the like. By elucidating actions of acompound on the upper gastrointestinal tract and renal functions usingone or more of these methods, usefulness of the compound as an activeingredient of a medicament can be confirmed.

EXAMPLES

Hereafter, the present invention will be further specifically explainedwith reference to examples, reference examples, preparation examples,and test examples (these may be henceforth collectively referred to as“examples and the like”). However, the scope of the present invention isnot limited to the following examples and the like

In the examples and the like, for thin layer chromatography (TLC),Precoated Silica Gel 60 F254 (produced by Merck, product number5715-1M)) was used. After development with chloroform:methanol (1:0 to1:1), acetonitrile:acetic acid:water (200:1:1 to 100:4:4) or ethylacetate:hexane (1:0 to 0:1), confirmation was performed by UVirradiation (254 nm or 365 nm), or coloration with iodine solution,aqueous potassium permanganate, phosphomolybdic acid (ethanol solution)or the like.

For drying organic solvent, anhydrous magnesium sulfate or anhydroussodium sulfate was used.

For column chromatography, Multi Prep YFLC produced by YamazenCorporation, or 2-ch parallel purification apparatus “Purif-α2(50F)”produced by MORITEX Corporation was used. In the case of Multi PrepYFLC, any of Ultra Pack Si-40A, 40B and 40D produced by YamazenCorporation was used as the column, and in the case of Purif-α2(50F),PurifPack-Si series produced by MORITEX Corporation was used as thecolumn.

For flash column chromatography, Silica gel 60N (spherical shape,neutral, 40 to 100 μm, produced by Kanto Chemicals) was used.

Preparative thin layer chromatography (henceforth also referred to as“PTLC”) was performed by using one or several plates of PLC Plate SilicaGel 60 F254 (20×20 cm, layer thickness 2 mm, including concentrationzone (4 cm), produced by Merck, product number 13793-1M) were useddepending on the amount of sample.

For HPLC purification, a liquid chromatography preparation andpurification apparatus produced by Waters Japan was used, DevelosilC30-UG-5 (produced by Nomura Kagaku) or the like was used as the column,and water-acetonitrile solvent containing 0.1% acetic acid was used asthe eluent.

When purification was performed by HPLC, the object compound wasobtained by removing the solvent by lyophilization, unless particularlyindicated. For the measurement of nuclear magnetic resonance (NMR)spectra, Gemini-300 (FT-NMR, Varian Co., Ltd.) or AL-300 (FT-NMR,produced by JEOL Co., Ltd.) was used. As the solvent, deuteratedchloroform was used unless specifically indicated, chemical shifts weremeasured by using tetramethylsilane (TMS) as an internal standard, andindicated with δ (ppm), and the binding constant was indicated with J(Hz).

For LCMS, mass spectrum was measured by liquid chromatography-massspectrometry (LC-MS). Unless especially indicated, a single quadrupolemass spectrometer, UPLC/SQD System (produced by Waters) was used as themass spectrometer, and the measurement was performed by the electrosprayionization (ESI) method. As the liquid chromatography apparatus, AcquityUltra Performance LC System produced by Waters was used. As theseparation column, ACQUITY UPLC BEH C18 (1×50 mm, 1.7 μm, produced byWaters) was used.

However, for the LC conditions of FLC-1 mentioned below, a singlequadrupole mass spectrometer, Platform-LC (produced by Waters) was usedas the mass spectrometer, and the measurement was performed by theelectrospray ionization (ESI) method. As the liquid chromatographyapparatus, 306 PUMP System produced by GILSON was used. As theseparation column, Develosil C30-UG-5 (50×4.6 mm, produced by NomuraKagaku) was used.

When LC conditions are especially mentioned in the examples andreference examples, it means that the measurement was performed with thefollowing solvent conditions. The symbol m/z means mass spectrum data(MH⁺, M⁺NH⁴⁺, or MH⁻ is also indicated).

(LC-1) The measurement was performed under the conditions that theelution was performed at a flow rate of 0.6 ml/minute using a lineargradient of 5 to 90% (v/v) of Solution B [acetonitrile containing 0.1%(v/v) acetic acid] in Solution A [water containing 0.1% (v/v) aceticacid] from 0 minute to 2.0 minutes, and then a linear gradient of 90 to98% of Solution B in Solution A from 2.0 to 2.5 minutes.(LC-6) The measurement was performed under the conditions that theelution was performed at a flow rate of 0.6 ml/minute using a lineargradient of 70 to 90% (v/v) of Solution B [acetonitrile containing 0.1%(v/v) acetic acid] in Solution A [water containing 0.1% (v/v) aceticacid] from 0 to 2.0 minutes, and then a linear gradient of 90 to 98% ofSolution B in Solution A from 2.0 to 2.5 minutes.(NLC-1) The measurement was performed under the conditions that theelution was performed at a flow rate of 0.6 ml/minute using a lineargradient of 5 to 90% (v/v) of Solution B [acetonitrile] in Solution A[10 mM aqueous ammonium acetate] from 0 minute to 2.0 minutes, and thena linear gradient of 90 to 98% of Solution B in Solution A from 2.0 to2.5 minutes.(NLC-6) The measurement was performed under the conditions that theelution was performed at a flow rate of 0.6 ml/minute using a lineargradient of 70 to 90% (v/v) of Solution B [acetonitrile] in Solution A[10 mM aqueous ammonium acetate] from 0 minute to 2.0 minutes, and thena linear gradient of 90 to 98% of Solution B in Solution A from 2.0 to2.5 minutes.(FLC-1) The measurement was performed under the conditions that theelution was performed at a flow rate of 2 ml/minute using a lineargradient of 5 to 98% (v/v) of Solution B [acetonitrile containing 0.1%(v/v) acetic acid] in Solution A [water containing 0.1% (v/v) aceticacid] from 0 to 5 minutes, 98% (v/v) of Solution B in Solution A from 5to 6 minutes, a linear gradient of 98 to 5% (v/v) Solution B in SolutionA from 6 to 6.01 minutes, and 5% (v/v) of Solution B in Solution A from6.01 to 7.5 minutes.

For chiral LC, retention time was measured by high performance liquidchromatography (HPLC). When chiral LC conditions are especiallymentioned in the examples and reference examples, it means that themeasurement was performed with the following measurement conditions.

(Chiral LC-1) The measurement was performed by using CHIRALCEL OD-H(4.6×250 mm, 5 μm, produced by Daicel Corporation) as the separationcolumn under the conditions that the elution was performed at a flowrate of 0.6 ml/minute as isocratic elution using 5% (v/v) of Solution B(ethanol) in Solution A (n-hexane).(Chiral LC-2) The measurement was performed by using CHIRALCEL OJ-H(4.6×250 mm, 5 μm, produced by Daicel Corporation) as the separationcolumn under the conditions that the elution was performed at a flowrate of 1.0 ml/minute using ethanol containing 0.1% (v/v) oftrifluoroacetic acid.

The manufacturers of the regents used may sometimes be indicated withthe following abbreviations: Tokyo Chemical Industry Co., Ltd., TCI;Sigma-Aldrich Co. LLC., ALDRICH; Kanto Kagaku Co., Inc., KANTO; WakoPure Chemical Industries Ltd., WAKO; Maybridge Co., Ltd., MAYBRIDGE;APOLLO Co., Ltd., APOLLO; Combi-Blocks Inc., COMBI-BLOCKS; TakasagoInternational Corporation, TAKASAGO; Johnson Matthey Co., Ltd., JOHNSON;Nippon Chemical Industrial Co., Ltd., Nippon Chemical; and JapanEnviroChemicals, Limited, Japan EnviroChemicals.

The abbreviations or symbols used in the descriptions have the followingmeanings: n, normal; i, iso; s, secondary; t, tertiary; c, cyclo; Me,methyl; Et, ethyl; Pr, propyl; Bu, butyl; Pen, pentyl; Hex, hexyl; Hep,heptyl; Ph, phenyl; Bn, benzyl; Py, pyridyl; Ac, acetyl; CHO, formyl;COOH, carboxyl; NO₂, nitro; DMA, dimethylamino; NH₂, amino; CF₃,trifluoromethyl; F, fluoro; Cl, chloro; Br, bromo; OMe, methoxy; OH,hydroxy; TFA, trifluoroacetyl; SO₂, sulfonyl; CO, carbonyl; THF,tetrahydrofuran; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;and DME, dimethoxyethane.

The numbers indicated before the substituents represent substitutionpositions. The numbers indicated before the abbreviations of aromaticrings with hyphens indicate the substitution positions on the aromaticrings. The symbol (S) mentioned in the compound names and structuralformulas means that the corresponding asymmetric carbon is in theS-configuration, and (R) means that the corresponding asymmetric carbonis in the R-configuration. Further, when (R) or (S) is not indicated fora compound having an asymmetric carbon, it means that the compoundconsisted of a mixture of (R)-isomer and (S)-isomer at an arbitraryratio. Such a compound may be a racemic mixture of (R)-isomer and(S)-isomer.

When deprotection is required in the synthesis process of the examplecompounds, it was performed according to known methods such as themethods described in Protective Groups in Organic Synthesis, publishedby John Wiley and Sons (2007).

Reference Example A-2: tert-Butyldimethyl(2-(thiophen-2-yl)ethoxy)silane(Intermediate A-2)

To a solution of 2-(thiophen-2-yl)ethanol (4 g, TCI) inN,N-dimethylformamide (312 mL), imidazole (4.3 g),tert-butyldimethylchlorosilane (7.05 g), andN,N-dimethyl-4-aminopyridine (763 mg) were successively added under icecooling, and the mixture was stirred at room temperature for 2.75 hours.To the reaction mixture, ethyl acetate was added, and the organic layerwas successively washed with 1 mol/L hydrochloric acid, saturated brine,saturated aqueous sodium hydrogencarbonate, and saturated brine, andthen dried. The solvent was evaporated under reduced pressure to obtainthe title compound (7.32 g).

(Intermediate A-2: Rf (TLC)=0.70 (hexane:ethyl acetate=4:1))

Reference Example A-3:5-(2-((tert-Butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylic Acid(Intermediate A-3)

A solution of the intermediate A-2 (7.15 g) in tetrahydrofuran (111 mL)was cooled to −78° C. under a nitrogen atmosphere. To the reactionmixture, n-butyllithium (2.6 mol/L solution in hexane, 14.3 mL, KANTO)was added dropwise, and the mixture was stirred for 0.75 hour as it was.The reaction mixture was warmed to −5° C., then dry ice (125 g) wasadded portionwise, and after completion of the addition, the reactionmixture was further stirred for 0.75 hour. To the reaction mixture,saturated aqueous ammonium chloride was added, and the mixture wasstirred at room temperature. Ethyl acetate was added to the reactionmixture for extraction, and the organic layer was washed successivelywith saturated aqueous ammonium chloride, and saturated brine, and thendried. The solvent was evaporated under reduced pressure to obtain thetitle compound (9.03 g).

(Intermediate A-3: LCMS m/z 287.0 (MH⁺), retention time 1.35 minutes, LCconditions NLC-1)

Reference Example A-4: Methyl 5-(2-hydroxyethyl)thiophene-2-carboxylate(Intermediate A-4)

A solution of the intermediate A-3 (9.03 g) in methanol (64 mL) wascooled to 0° C., concentrated sulfuric acid (3.2 mL) was addedportionwise to the solution, and the mixture was stirred as it was for 5minutes. The reaction mixture was heated to 70° C., stirred for 24hours, and then cooled to 0° C., and saturated aqueous sodiumhydrogencarbonate was added portionwise until the reaction mixturebecame neutral. Ethyl acetate was added to the reaction mixture forextraction, and the organic layer was washed successively with saturatedaqueous sodium hydrogencarbonate, and saturated brine, and then dried.The solvent was evaporated under reduced pressure, and the residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (4.61 g).

(Intermediate A-4: Rf (TLC)=0.33 (hexane:ethyl acetate=1:1))

Reference Example A-5: Methyl 5-(2-bromoethyl)thiophene-2-carboxylate(Intermediate A-5)

To a solution of the intermediate A-4 (4.61 g) in dichloromethane (200mL), triphenylphosphine (9.8 g) was added, and then the mixture wascooled to 0° C. To the reaction mixture, carbon tetrabromide (12.3 g)was added portionwise, and the mixture was warmed to room temperature,and then stirred for 13.5 hours. The reaction mixture was decompressedto evaporate the solvent, and then the residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (5.4g).

(Intermediate A-5: Rf (TLC)=0.70 (hexane:ethyl acetate=1:1))

Reference Example A-6: tert-Butyl2-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate(Intermediate A-6)

To a solution of the intermediate A-5 (6.2 g) in acetonitrile (125 mL),tert-butyl carbazate (16.5 g, TCI), sodium hydrogencarbonate (10.5 g),and sodium iodide (700 mg) were successively added, and the mixture wasstirred at 90° C. for 13 hours. The reaction mixture was cooled to 0°C., ethyl acetate (155 mL) was added to the reaction mixture, and theorganic layer was successively washed with 1 mol/L hydrochloric acid,saturated aqueous sodium hydrogencarbonate, and saturated brine, andthen dried. The solvent was evaporated under reduced pressure, and theresidue was purified by column chromatography (hexane/ethyl acetate) toobtain the title compound (5.1 g).

(Intermediate A-6: LCMS m/z 301.1 (MH⁺), retention time 1.42 minutes, LCconditions NLC-1)

¹H-NMR (CDCl₃): δ (ppm) 7.64 (1H, d, J=4.0 Hz), 6.87 (1H, d, J=4.0 Hz),3.86 (3H, s), 3.18 (2H, t, J=7.2 Hz), 3.02 (2H, t, J=7.2 Hz), 2.60-1.90(2H, br), 1.64 (9H, s)

Reference Example B-3: S-(2-chloroethyl)carbochloride Thioate(Intermediate B-3)

A mixed solution of ethylene sulfide (320 g, TCI), and pyridine (4.3 mL)was cooled on an ice bath under an argon atmosphere, triphosgene (474 g,TCI) was added portionwise to the reaction mixture, and the mixture wasstirred as it was for 4 hours. The reaction mixture was distilled underreduced pressure (0.7 to 0.8 kPa, 50 to 52° C.) for purification toobtain the title compound (281 g).

¹H-NMR (CDCl₃): δ (ppm) 3.72 (2H, t, J=7.0 Hz), 3.30 (2H, t, J=7.0 Hz)

Reference Example Z-1: tert-Butyl2-(((2-chloroethyl)thio)carbonyl)-2-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate(Intermediate Z-1)

To a solution of the intermediate A-6 (140.3 g) in dichloromethane (660mL), water (330 mL), and sodium hydrogencarbonate (78.09 g) were added,the mixture was stirred for 10 minutes, and then the intermediate B-3(81.71 g) was added portionwise to the reaction mixture, while theinternal temperature of the reaction mixture was maintained to be at 20to 25° C. The mixture was stirred as it was for 1 hour, and then theorganic layer was washed with saturated brine, and dried. The solventwas evaporated under reduced pressure to obtain the title compound(199.5 g).

(Intermediate Z-1: Rf (TLC)=0.43 (hexane:ethyl acetate=2:1))

Reference Example Z-2: Methyl5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate(Intermediate Z-2)

To the intermediate Z-1 (199 g), a 4 mol/L solution of hydrogen chloridein dioxane (800 mL) was added, and the mixture was stirred at roomtemperature for 18 hours. The solvent was evaporated under reducedpressure, and then dichloromethane (6 L), and saturated aqueous sodiumhydrogencarbonate (2 L) were added to the residue for extraction. Theorganic layer was washed with saturated brine (2 L), and dried, and thenthe solvent was evaporated under reduced pressure to obtain the titlecompound (172 g).

(Intermediate A-4: Rf (TLC)=0.49 (heptane:ethyl acetate=1:1))

Reference Example Z-3: Methyl5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-3)

To a solution of the intermediate Z-2 (172 g) in acetonitrile (3.4 L),sodium hydrogencarbonate (223 g), and sodium iodide (397 g) weresuccessively added, and the mixture was stirred at 75° C. for 15 hours.The mixture was further stirred at 83° C. for 15 hours, and then cooledto room temperature. The reaction mixture was filtered by using filterpaper, and the residue remained on the filter paper was washed withacetonitrile (1 L), combined with the filtrate, and concentrated underreduced pressure. To the residue obtained after the concentration,dichloromethane (3 L) was added, and then the mixture was filtered byusing filter paper, the residue remained on the filter paper was washedwith dichloromethane (1 L), combined with the filtrate, and concentratedunder reduced pressure. The resulting residue was purified by columnchromatography (hexane/ethyl acetate), and then concentrated underreduced pressure. The residue was dissolved in ethyl acetate (1 L) bywarming, then heptane was added to the solution, and the mixture wascooled on ice. The deposited solid was collected by filtration to obtainthe title compound (97 g).

¹H-NMR (CDCl₃): δ (ppm) 7.64 (1H, d, J=3.8 Hz), 6.88 (1H, d, J=3.8 Hz),3.86 (3H, s), 3.85 (2H, t, 7.0 Hz), 3.30 (2H, t, J=7.0 Hz), 3.25-3.17(4H, m), 3.16 (2H, t, J=7.0 Hz)

Reference Example C-2: 2-(3-Bromophenyl)-N-methoxy-N-methylacetamide(Intermediate C-2)

A solution of diisopropylethylamine (800 mL) in dichloromethane (1.8 L)was cooled on ice, 3-bromophenylacetic acid (313 g, TCI),N,O-dimethylhydroxylamine hydrochloride (284 g),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (334 g), andN,N-dimethyl-4-aminopyridine (18 g) were successively added, and themixture was stirred at room temperature for 12.5 hours. To the reactionmixture, water (630 mL), and dichloromethane (630 mL) were added, andthen the organic layer was successively washed twice with 2 mol/Lhydrochloric acid (630 mL), and once each with saturated aqueous sodiumhydrogencarbonate (630 mL), and saturated brine (630 mL). The organiclayer was dried, and then the solvent was evaporated under reducedpressure to obtain the title compound (372 g).

(Intermediate C-2: LCMS m/z 257.9 (MH⁺), retention time 1.37 minutes, LCconditions NLC-1)

¹H-NMR (CDCl₃): δ (ppm) 7.46-7.44 (1H, m), 7.39-7.36 (1H, m), 7.25-7.15(2H, m), 3.74 (2H, s), 3.64 (3H, s), 3.20 (3H, s)

Reference Example C-3: 1-(3-Bromophenyl)but-3-en-2-one (IntermediateC-3)

A solution of the intermediate C-2 (104.2 g) in tetrahydrofuran (2.1 L)was cooled to −45° C. under a nitrogen atmosphere. To the reactionmixture, vinylmagnesium bromide (1 mol/L solution in tetrahydrofuran,605 mL, Aldrich) was added over 30 minutes, and the mixture was warmedto 0° C., and then stirred for 1.5 hours. The reaction mixture was addedto a mixture of ice water (1 L), and 2 mol/L hydrochloric acid (1 L),and the mixture was stirred for 1 minute. Isopropyl ether (2 L) wasadded for extraction, and the organic layer was successively washed with1 mol/L hydrochloric acid (1 L), water (1 L), and saturated brine (1 L),and dried. The solvent was evaporated under reduced pressure to obtainthe title compound (92.1 g).

(Intermediate C-3: Rf (TLC)=0.74 (heptane:ethyl acetate=2:1))

Reference Example C-2-2: 2-(3-Iodophenyl)-N-methoxy-N-methylacetamide(Intermediate C-2-2)

The intermediate C-2-2 was synthesized according to the method describedin Reference Example C-2 by using 3-iodophenylacetic acid (2.85 g)instead of 3-bromophenylacetic acid, and thus the title compound (3.07g) was obtained.

(Intermediate C-2-2: Rf (TLC)=0.42 (hexane:ethyl acetate=1:2))

When the compound is synthesized according to the method describedabove, amount of the reagents, amount of the solvent, reaction time andthe like can be appropriately changed according to the equivalent amountof the starting material to be used in light of common knowledge ofthose skilled in the art. The same shall apply to the followingexamples.

Reference Example C-3-2: 1-(3-Iodophenyl)but-3-en-2-one (IntermediateC-3-2)

The intermediate C-3-2 was synthesized according to the method describedin Reference Example C-3 by using the intermediate C-2-2 (100 mg)instead of the intermediate C-2, and thus the title compound (58.7 mg)was obtained.

(Intermediate C-3-2: Rf (TLC)=0.60 (hexane:ethyl acetate=1:2))

Reference Example Z-4: Methyl5-(2-(4-(4-(3-bromophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4)

To a solution of the intermediate Z-3 (44.4 g) in ethanol (444 mL), theintermediate C-3 (92.1 g) was added, and the mixture was stirred at 110°C. for 40 hours. The reaction mixture was decompressed to evaporate thesolvent. The resulting residue was purified by column chromatography(toluene/ethyl acetate) to obtain the title compound (75.9 g).

(Intermediate Z-4: LCMS m/z 511.2 (MH⁺), retention time 1.75 minutes, LCconditions NLC-1)

Reference Example Z-4-2: Methyl5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4-2)

The intermediate Z-4-2 was synthesized according to the method describedin Reference Example Z-4 by using the intermediate C-3-2 (680.2 mg)instead of the intermediate C-3, and thus the title compound (120.1 mg)was obtained.

(Intermediate Z-4-2: Rf (TLC)=0.50 (hexane:ethyl acetate=1:2), LCMS m/z559.0 (MH⁺), retention time 1.84 minutes, LC conditions LC-1)

Reference Example Z-5: Methyl5-(2-(4-(4-(3-bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-5)

A solution of the intermediate Z-4 (75.7 g) in methanol (1.14 L) wascooled to 0° C., and sodium borohydride (7.47 g) was added portionwiseto the solution. The mixture was stirred at 0° C. for 1 hour, and thendiluted hydrochloric acid was added portionwise to the reaction mixtureuntil the reaction mixture became neutral. The organic solvent wasevaporated under reduced pressure, then ethyl acetate (2 L) was added tothe residue, the mixture was cooled to 0° C., saturated aqueous sodiumhydrogencarbonate (1 L) was added to the mixture portionwise, and theresulting mixture was stirred for 5 minutes. The organic layer wasextracted, and dried, and then the solvent was evaporated under reducedpressure to obtain the title compound (71.0 g).

(Intermediate Z-5: LCMS m/z 513.15 (MH⁺), retention time 1.70 minutes,LC conditions LC-1)

Reference Example Z-14: Methyl5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-14)

To the intermediate Z-4-2 (86.6 mg), diethylamine (78 μL),3-ethynylthiophene (21 μL), copper(I) iodide (0.8 mg), andtetrakis(triphenylphosphine)palladium (1.1 mg) were successively added,and the mixture was stirred at room temperature for 2 hours. Further,diethylamine (600 μL), copper(I) iodide (1.5 mg), andtetrakis(triphenyl-phosphine)palladium (2.0 mg) were successively added,and the mixture was stirred at room temperature for 12 hours. To thereaction mixture, diethyl ether, and 1 mol/L hydrochloric acid (0.5 mL)were added, and the organic layer was successively washed 5 times with 1mol/L hydrochloric acid (1 mL), and once with saturated aqueous sodiumhydrogencarbonate (0.5 mL), and dried. The solvent was evaporated underreduced pressure, and the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (31.7mg).

(Intermediate Z-14: Rf (TLC)=0.12 (hexane:ethyl acetate=1:2), LCMS m/z539.1 (MH⁺), retention time 1.95 minutes, LC conditions LC-1)

Reference Example Z-17: Methyl5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-17)

The intermediate Z-17 was synthesized according to the method describedin Reference Example Z-5 by using the intermediate Z-14 (31.7 mg)instead of the intermediate Z-4, and thus the title compound (31.8 mg)was obtained.

(Intermediate Z-17: LCMS m/z 541.1 (MH⁺), retention time 1.90 minutes,LC conditions LC-1)

Example 1:5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate Z-17 (31.8 mg) in tetrahydrofuran (884μL), water (221 μL), and 2 mol/L aqueous lithium hydroxide (442 μL) wereadded, and the mixture was stirred at 50° C. for 17.5 hours. Thereaction mixture was cooled to 0° C., 2 mol/L hydrochloric acid (660 μL)was added to the mixture, and then the resulting mixture was extractedwith ethyl acetate. The solvent was evaporated under reduced pressure,and the resulting residue was purified by thin layer chromatography(hexane/ethyl acetate) to obtain the title compound (22.7 mg).

(LCMS m/z 527.2 (MH⁺), retention time 1.68 minutes, LC conditions LC-1)

Reference Example A-10: (3-Bromothiophen-2-yl)methanol (IntermediateA-10)

A solution of 3-bromothiophene-2-carboxylic acid (3.0 g, Aldrich) intetrahydrofuran (46 mL) was cooled to 0° C. under a nitrogen gasatmosphere, a 1 mol/L solution of borane-tetrahydrofuran complex intetrahydrofuran (26.1 mL) was added dropwise to the solution over 15minutes, and then the mixture was stirred at room temperature for 21.5hours. The reaction mixture was cooled to 0° C., ice water, 1 mol/Lhydrochloric acid, and ethyl acetate were added to the mixture, and theresulting mixture was stirred. The organic solvent was evaporated underreduced pressure, then ethyl acetate was added to the residue, and theorganic layer was successively washed with 1 mol/L hydrochloric acid,saturated aqueous sodium hydrogencarbonate, and saturated brine, anddried. The solvent was evaporated under reduced pressure to obtain thetitle compound (2.87 g).

(Intermediate A-10: Rf (TLC)=0.42 (hexane:ethyl acetate=2:1))

Reference Example A-11: 3-Bromo-2-(bromomethyl)thiophene (IntermediateA-11)

A solution of the intermediate A-10 (7.14 g) in dichloromethane (169 mL)was cooled to 0° C., triphenylphosphine (13.3 g), and carbontetrabromide (13.45 g) were added to the solution, and the mixture wasstirred at room temperature for 2.75 hours. To the reaction mixture,saturated aqueous sodium hydrogencarbonate was added, and then theorganic solvent was evaporated under reduced pressure. Ethyl acetate wasadded the residue, and then the organic layer was successively washedwith saturated aqueous sodium hydrogencarbonate, and saturated brine,and dried. The organic solvent was evaporated under reduced pressure,then a mixed solvent of hexane and ethyl acetate (8:1) was added to theresulting residue to prepare a suspension, and the suspension wasfiltered with filter paper covered with silica gel. The solvent of thefiltrate was evaporated under reduced pressure to obtain the titlecompound (9.70 g).

(Intermediate A-11: Rf (TLC)=0.64 (hexane:ethyl acetate=8:1))

Reference Example A-12: 2-(3-Bromothiophen-2-yl)acetonitrile(Intermediate A-12)

To the intermediate A-11 (9.70 g), dimethyl sulfoxide (28 mL), andacetonitrile (140 mL) were added, the mixture was cooled to 0° C., thensodium cyanide (2.15 g) was added to the mixture, and the resultingmixture was stirred at room temperature for 16 hours. To the reactionmixture, saturated aqueous sodium hydrogencarbonate was added, and themixture was stirred, and then concentrated under reduced pressure. Thereaction mixture was filtered through filter paper covered with Celite,and the residue remained on Celite was washed with ethyl acetate. Thefiltrate and the wash liquid were mixed, and the organic layer wassuccessively washed with saturated aqueous sodium hydrogencarbonate, andsaturated brine, and dried. The organic solvent was evaporated underreduced pressure, and then the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (3.89g).

(Intermediate A-12: Rf (TLC)=0.18 (hexane:ethyl acetate=8:1))

Reference Example A-13: Ethyl 2-(3-bromothiophen-2-yl)acetate(Intermediate A-13)

To a solution of the intermediate A-12 (3.89 g) in ethanol (32.3 mL),water (0.4 mL) was added, the mixture was cooled to 0° C., and thenconcentrated sulfuric acid (5.63 mL) was added to the mixtureportionwise. The reaction mixture was stirred at 85° C. for 115 hours,and then cooled to 0° C., and saturated aqueous sodium hydrogencarbonatewas added until the reaction mixture became neutral. Ethyl acetate wasadded to the reaction mixture, and the resulting mixture was stirred,and then concentrated under reduced pressure. Ethyl acetate was added tothe reaction mixture, and the organic layer was successively washed withsaturated aqueous sodium hydrogencarbonate, and saturated brine, anddried. The solvent was evaporated under reduced pressure to obtain thetitle compound (4.40 g).

(Intermediate A-13: Rf (TLC)=0.33 (hexane:ethyl acetate=8:1))

Reference Example A-14: 2-(3-Bromothiophen-2-yl)ethanol (IntermediateA-14)

A solution of the intermediate A-13 (4.40 g) in tetrahydrofuran (88.5mL) was cooled to 0° C. under a nitrogen gas atmosphere, lithiumaluminum hydride (672 mg) was added to the solution, and the mixture wasstirred for 0.6 hour. To the reaction mixture, ice water, 1 mol/Lhydrochloric acid, and ethyl acetate were added, and the resultingmixture was stirred, and then the organic layer was successively washedwith 1 mol/L hydrochloric acid, saturated aqueous sodiumhydrogencarbonate, and saturated brine, and dried. The solvent wasevaporated under reduced pressure, and the resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (2.69 g).

(Intermediate A-14: Rf (TLC)=0.23 (hexane:ethyl acetate=4:1))

Reference Example A-2-2:(2-(3-Bromothiophen-2-yl)ethoxy)(tert-butyl)dimethylsilane (IntermediateA-2-2)

The intermediate A-2-2 was synthesized according to the method describedin Reference Example A-2 by using the intermediate A-14 (2.69 g) insteadof 2-(thiophen-2-yl)ethanol, and thus the title compound (3.93 g) wasobtained.

(Intermediate A-2-2: Rf (TLC)=0.76 (hexane:ethyl acetate=4:1))

Reference Example A-3-2:4-Bromo-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylicAcid (Intermediate A-3-2)

The intermediate A-3-2 was synthesized according to the method describedin Reference Example A-3 by using the intermediate A-2-2 (293 mg)instead of the intermediate A-2, and lithium diisopropylamide (1.09mol/L solution in hexane/tetrahydrofuran, 928 μL, KANTO) instead ofn-butyllithium (2.6 mol/L solution in hexane, KANTO), and thus the titlecompound (339 mg) was obtained.

(Intermediate A-3-2: Rf (TLC)=0.12 (hexane:ethyl acetate=1:1))

Reference Example A-4-2: Methyl4-bromo-5-(2-hydroxyethyl)thiophene-2-carboxylate (Intermediate A-4-2)

The intermediate A-4-2 was synthesized according to the method describedin Reference Example A-4 by using the intermediate A-3-2 (377 mg)instead of the intermediate A-3, and thus the title compound (217 mg)was obtained.

(Intermediate A-4-2: Rf (TLC)=0.53 (hexane:ethyl acetate=1:1))

Reference Example A-5-2: Methyl4-bromo-5-(2-bromoethyl)thiophene-2-carboxylate (Intermediate A-5-2)

The intermediate A-5-2 was synthesized according to the method describedin Reference Example A-5 by using the intermediate A-4-2 (217 mg)instead of the intermediate A-4, and thus the title compound (315 mg)was obtained.

(Intermediate A-5-2: Rf (TLC)=0.44 (hexane:ethyl acetate=8:1))

Reference Example A-6-2: tert-Butyl2-(2-(3-bromo-5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate(Intermediate A-6-2)

The intermediate A-6-2 was synthesized according to the method describedin Reference Example A-6 by using the intermediate A-5-2 (315 mg)instead of the intermediate A-5, and thus the title compound (168 mg)was obtained.

(Intermediate A-5-2: Rf (TLC)=0.48 (hexane:ethyl acetate=1:1))

Reference Example Z-1-2: tert-Butyl2-(2-(3-bromo-5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-(((2-chloroethyl)thio)carbonyl)hydrazinecarboxylate(Intermediate Z-1-2)

To a solution of the intermediate A-6-2 (1.98 g) in dichloromethane(13.1 mL), sodium hydrogencarbonate (880 mg) was added, the mixture wascooled to 0° C., and then the intermediate B-3 (993 mg) was addedportionwise to the mixture. The resulting mixture was stirred at roomtemperature for 0.5 hour, then water and ethyl acetate were added to thereaction mixture, and the organic layer was successively washed withsaturated aqueous sodium hydrogencarbonate, and saturated brine, anddried. The solvent was evaporated under reduced pressure to obtain thetitle compound (796 mg).

(Intermediate Z-1-2: Rf (TLC)=0.53 (toluene:ethyl acetate=8:1))

Reference Example Z-2-2: Methyl4-bromo-5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate(Intermediate Z-2-2)

To the intermediate Z-1-2 (796 mg), a 4 mol/L solution of hydrogenchloride in dioxane (7.5 mL) was added, and the mixture was stirred atroom temperature for 17.6 hours. To the reaction mixture, ethyl acetate,and 5 mol/1 aqueous sodium hydroxide were added, and then saturatedaqueous sodium hydrogencarbonate was added to the mixture until themixture became basic. The organic layer was successively washed withsaturated aqueous sodium hydrogencarbonate, and saturated brine, anddried, and then the solvent was evaporated under reduced pressure toobtain the title compound (594 mg).

(Intermediate Z-2-2: Rf (TLC)=0.42 (toluene:ethyl acetate=8:1))

Reference Example Z-3-2: Methyl4-bromo-5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-3-2)

To a solution of the intermediate Z-2-2 (594 mg) in acetonitrile (14.9mL), sodium hydrogencarbonate (626 mg), and sodium iodide (1.12 g) weresuccessively added, and the mixture was stirred at 85° C. for 120 hours.The reaction mixture was cooled to room temperature, and then ethylacetate, and water were added to the mixture for extraction. The organiclayer was successively washed with saturated aqueous sodiumhydrogencarbonate, and saturated brine, and dried, then the solvent wasevaporated under reduced pressure, and the resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (374 mg).

(Intermediate Z-3-2: Rf (TLC)=0.13 (hexane:ethyl acetate=2:1))

Reference Example C-4:N-Methoxy-N-methyl-2-(3-(thiophen-3-ylethynyl)-phenyl)acetamide(Intermediate C-4)

To a solution of the intermediate C-2-2 (1.0 g) in acetonitrile (26 mL),bis(acetonitrile)palladium chloride (43 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (243 mg), cesiumcarbonate (2.1 g), and 3-ethynylthiophene (650 μL) were successivelyadded, and the mixture was stirred at 60° C. for 14 hours under anitrogen gas atmosphere. The reaction mixture was filtered throughfilter paper covered with Celite, and the residue remained on Celite waswashed with ethyl acetate. The filtrate and the wash liquid were mixed,the organic solvent was evaporated under reduced pressure, and then theresulting residue was purified by column chromatography (hexane/ethylacetate) to obtain the title compound (850 mg).

(Intermediate C-4: Rf (TLC)=0.40 (hexane:ethyl acetate=1:1), LCMS m/z286.13 (MH⁺), retention time 1.70 minutes, LC conditions LC-1)

Reference Example Z-14-2: Methyl4-bromo-5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-14-2)

A solution of the intermediate C-4 (117 mg) in 1,2-dimethoxyethane (2.3mL) was cooled to 0° C. under a nitrogen atmosphere. To the reactionmixture, vinylmagnesium bromide (1 mol/L solution in tetrahydrofuran,620 μL, Aldrich) was added, and the resulting mixture was stirred for 3hours. To the reaction mixture, 2 mol/L hydrochloric acid was added, andthe resulting mixture was stirred for 1 minute. Ethyl acetate was addedto the reaction mixture for extraction, the organic layer was dried, andthen the solvent was evaporated under reduced pressure. To the resultingresidue, ethanol (3 mL), water (3 mL), and the intermediate Z-3-2 (100mg) were added, and the mixture was stirred at 110° C. for 18 hours. Tothe reaction mixture, saturated brine, and chloroform were added forextraction, the organic layer was dried, and then the solvent wasevaporated under reduced pressure. The resulting residue was purified bycolumn chromatography (hexane/ethyl acetate) to obtain the titlecompound (156.1 mg).

(Intermediate Z-14-2: LCMS m/z 617.2 (MH⁺), retention time 2.08 minutes,LC conditions LC-1)

Example 2:4-Bromo-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-14-2 (156.1 mg) in methanol (3 mL) wascooled to 0° C., and sodium borohydride (17.3 mg) was added portionwiseto the solution. The resulting mixture was stirred at 0° C. for 1 hour,and then diluted hydrochloric acid was added portionwise to the mixtureuntil the reaction mixture became neutral. The organic solvent wasevaporated under reduced pressure, then ethyl acetate (2 L) was added tothe residue, the mixture was cooled to 0° C., saturated aqueous sodiumhydrogencarbonate (1 L) was added portionwise to the mixture, and theresulting mixture was stirred for 5 minutes. The organic layer wasextracted, and dried, and then the solvent was evaporated under reducedpressure. To the resulting residue, tetrahydrofuran (4.6 mL), methanol(4.6 mL), and 1 mol/L aqueous sodium hydroxide (4.6 mL) were added, andthe mixture was stirred at room temperature for 3 hours. The reactionmixture was cooled to 0° C., 2 mol/L hydrochloric acid was added to thereaction mixture, then the resulting mixture was extracted with ethylacetate, and the organic layer was dried. The solvent was evaporatedunder reduced pressure, and the resulting residue was purified by columnchromatography (chloroform/methanol) to obtain the title compound (140mg).

(LCMS m/z 605.1 (MH⁺), retention time 1.78 minutes, LC conditions LC-1)

Reference Example Z-14-3: Methyl5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-2-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-14-3)

The intermediate Z-14-3 was synthesized according to the methoddescribed in Reference Example Z-14 by using 2-ethynylthiophene (33.6mg, MAYBRIDGE) instead of 3-ethynylthiophene, and thus the titlecompound (32.9 mg) was obtained.

(Intermediate Z-14-3: LCMS m/z 539.0 (MH⁺), retention time 2.00 minutes,LC conditions LC-1)

Example 3:5-(2-(4-(3-Hydroxy-4-(3-(thiophen-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-14-3 (32.9 mg) in methanol (0.6 mL) wascooled to 0° C., and sodium borohydride (3.6 mg) was added portionwiseto the solution. The mixture was stirred at 0° C. for 1.5 hours, andthen diluted with ethyl acetate, and diluted hydrochloric acid (1.5 mL)was added to the mixture. Saturated aqueous sodium hydrogencarbonate wasadded to the mixture until the mixture became neutral. The resultingmixture was extracted with ethyl acetate, the organic layer was dried,and then the solvent was evaporated under reduced pressure. To theresulting residue, tetrahydrofuran (920 μL), water (230 μL), and 2 mol/Laqueous sodium hydroxide (460 μL) were added, and the resulting mixturewas stirred at 50° C. for 14 hours. The reaction mixture was cooled to0° C., and left standing for 5.5 hours, then 2 mol/L hydrochloric acidwas added to the mixture, the mixture was extracted with ethyl acetate,and the organic layer was dried. The solvent was evaporated underreduced pressure, and the resulting residue was purified by liquidchromatography (acetonitrile/water) to obtain the title compound (3.6mg).

(LCMS m/z 527.0 (MH⁺), retention time 1.79 minutes, LC conditions LC-1)

Reference Example Z-6: Methyl5-(2-(4-(4-(3-bromophenyl)-3-((tert-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-6)

To a solution of the intermediate Z-5 (1.0 g) in N,N-dimethylformamide(19.5 mL), imidazole (265 mg), and tert-butyldimethylchlorosilane (596mg) were added, and the resulting mixture was stirred at 30° C. for 15hours. To the reaction mixture, saturated aqueous sodiumhydrogencarbonate was added, and the resulting mixture was extractedwith ethyl acetate. The organic layer was washed with saturated brine,and then dried, the solvent was evaporated under reduced pressure, andthe resulting residue was purified by column chromatography(hexane/ethyl acetate) to obtain the title compound (1.16 g).

(Intermediate Z-6: LCMS m/z 627.0 (MH⁺), retention time 2.53 minutes, LCconditions LC-1)

Reference Example Z-21: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((trimethylsilyl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-21)

To a solution of the intermediate Z-6 (300 mg) in acetonitrile (15.3mL), bis(acetonitrile)palladium chloride (12.4 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (68.3 mg), cesiumcarbonate (311 mg), and ethynyltrimethylsilane (331 μL) weresuccessively added, and the resulting mixture was stirred at 60° C. for19 hours under a nitrogen gas atmosphere. To the reaction mixture,ethynyltrimethylsilane (199 μL), bis(acetonitrile)palladium chloride(6.2 mg), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (34.1mg), and cesium carbonate (187 mg) were added, and the resulting mixturewas stirred at 60° C. for 3.75 hours. The solvent of the reactionmixture was evaporated under reduced pressure, and then the resultingresidue was purified by column chromatography (hexane/ethyl acetate) toobtain the title compound (245 mg).

(Intermediate Z-21: LCMS m/z 645.4 (MH⁺), retention time 2.35 minutes,LC conditions LC-6)

Reference Example Z-22: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-ethynylphenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-22)

To a solution of the intermediate Z-21 (225 mg) in methanol (3.6 mL),potassium carbonate (50 mg) was added, and the mixture was stirred atroom temperature for 1 hour. The reaction mixture was filtered, and thefiltrate was combined with the methanol wash liquid used for washing theresidue remained on the filter paper, and concentrated. The resultingresidue was purified by column chromatography (hexane/ethyl acetate) toobtain the title compound (198 mg).

(Intermediate Z-22: LCMS m/z 573.3 (MH⁺), retention time 1.37 minutes,LC conditions LC-6)

Example 4:5-(2-(4-(3-Hydroxy-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-4)

To a solution of the intermediate Z-22 (10 mg) in acetonitrile (280 μV,bis(acetonitrile)palladium chloride (0.5 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (2.5 mg), cesiumcarbonate (6.8 mg), and 3-bromo-4-methylthiophene (9.3 mg, TCI) weresuccessively added, and the resulting mixture was stirred at 60° C. for4 hours under a nitrogen gas atmosphere. The reaction mixture wasfiltered through filter paper covered with Celite, and the residueremained on Celite was washed with ethyl acetate. The filtrate and thewash liquid were mixed, and the organic layer was successively washedwith water, and saturated brine, and then dried. The solvent wasevaporated under reduced pressure, and then the resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (7.4 mg).

(Intermediate Z-7-4: LCMS m/z 699.4 (MH⁺), retention time 2.18 minutes,LC conditions LC-6)

[Step b]

5-(2-(4-(3-Hydroxy-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-4 (7.4 mg) in tetrahydrofuran (390μL) was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solutionin tetrahydrofuran, 33 μL) was added to the solution, and the resultingmixture was stirred at room temperature for 2.5 hours. To the reactionmixture, methanol (390 μl), and 1 mol/L aqueous sodium hydroxide (390μL) were added, and the resulting mixture was stirred at roomtemperature for 2 hours. To the reaction mixture, 2 mol/L hydrochloricacid (100 μL), and water (400 μL) were added, the mixture was extracted5 times with ethyl acetate (1 mL), and then the organic layer was dried.The solvent was evaporated under reduced pressure, and the resultingresidue was purified by column chromatography (chloroform/methanol) toobtain the title compound (4.9 mg).

(LCMS m/z 541.2 (MH⁺), retention time 1.74 minutes, LC conditions LC-1)

Example 5:5-(2-(4-(4-(3-((2-Chlorothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

According to the method described in Example 4, synthesis was performedby using 3-bromo-2-chlorothiophene (20.7 mg, TCI) instead of3-bromo-4-methylthiophene to obtain the title compound (12.9 mg).

(LCMS m/z 561.1 (MH⁺), retention time 1.77 minutes, LC conditions LC-1)

Example 6:5-(2-(4-(3-Hydroxy-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-6)

To a solution of the intermediate Z-22 (20 mg) in acetonitrile (1120μL), bis(acetonitrile)palladium chloride (0.9 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (5.0 mg), cesiumcarbonate (13.7 mg), and 3-bromo-5-methylthiophene (18.5 mg, TCI) weresuccessively added, and the resulting mixture was stirred at 60° C. for4 hours under a nitrogen gas atmosphere. The reaction mixture wasfiltered through filter paper covered with Celite, and the residueremained on Celite was washed with a mixed solvent of chloroform andmethanol (9:1). The filtrate and the wash liquid were mixed, the solventwas evaporated under reduced pressure, and then the resulting residuewas purified by column chromatography (hexane/ethyl acetate) to obtainthe title compound (16.2 mg).

(Intermediate Z-7-6: LCMS m/z 699.4 (MH⁺), retention time 2.20 minutes,LC conditions LC-6)

[Step b]

5-(2-(4-(3-Hydroxy-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-6 (16.2 mg) in tetrahydrofuran (850μL) was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solutionin tetrahydrofuran, 73 μL) was added, and the mixture was stirred atroom temperature for 2.5 hours. To the reaction mixture, 1 mol/L aqueoussodium hydroxide (66 μL) was added, and the resulting mixture wasstirred at room temperature for 5 hours. To the reaction mixture, 1mol/L hydrochloric acid (500 μL) was added, the resulting mixture wasextracted 5 times with ethyl acetate (1 mL), and then the organic layerwas washed with saturated brine (500 μl), and dried. The solvent wasevaporated under reduced pressure to obtain the title compound (22.1mg).

(LCMS m/z 541.2 (MH⁺), retention time 1.76 minutes, LC conditions LC-1)

Example 7:5-(2-(4-(4-(3-((4-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 4-bromothiophene-3-carbonitrile (18.9 mg, COMBI-BLOCKS) insteadof 3-bromo-5-methylthiophene to obtain the title compound (3.8 mg).

(LCMS m/z 552.1 (MH⁺), retention time 1.52 minutes, LC conditions LC-1)

Example 8:5-(2-(4-(4-(3-((2-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 3-bromothiophene-2-carbonitrile (18.9 mg, APOLLO) instead of3-bromo-5-methylthiophene to obtain the title compound (5.5 mg).

(LCMS m/z 552.2 (MH⁺), retention time 1.56 minutes, LC conditions LC-1)

Example 9:5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 4-bromothiazole (17.2 mg, ALDRICH) instead of3-bromo-5-methylthiophene to obtain the title compound (14.3 mg).

(LCMS m/z 528.2 (MH⁺), retention time 1.38 minutes, LC conditions LC-1)

Example 10:5-(2-(4-(4-(3-(Furan-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 3-bromofuran (15.4 mg, TCI) instead of3-bromo-5-methylthiophene to obtain the title compound (13.2 mg).

(LCMS m/z 511.2 (MH⁺), retention time 1.57 minutes, LC conditions LC-1)

Example 11:5-(2-(4-(4-(3-(Furan-2-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 2-bromofuran (14.8 mg, ALDRICH) instead of3-bromo-5-methylthiophene to obtain title compound (4.5 mg).

(LCMS m/z 511.2 (MH⁺), retention time 1.58 minutes, LC conditions LC-1)

Example 12:5-(2-(4-(4-(3-((5-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

Methyl5-(2-(4-(3-((tert-Butyldimethylsilyl)oxy)-4-(3-((5-cyanothiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-12)

To a solution of the intermediate Z-22 (14 mg) in acetonitrile (400 μL),bis(acetonitrile)palladium chloride (0.7 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (3.6 mg), cesiumcarbonate (12.3 mg), and 4-bromothiophene-2-carbonitrile (18.9 mg,COMBI-BLOCKS) were successively added, and the resulting mixture wasstirred at 60° C. for 6 hours under a nitrogen gas atmosphere. Thesolvent was evaporated under reduced pressure, and then the resultingresidue was purified by column chromatography (hexane/ethyl acetate) toobtain the title compound (6.4 mg).

(Intermediate Z-7-12: LCMS m/z 680.4 (MH⁺), retention time 1.71 minutes,LC conditions LC-6)

[Step b]

5-(2-(4-(4-(3-((5-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-12 (6.4 mg) in tetrahydrofuran (330μL) was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solutionin tetrahydrofuran, 28 μL) was added to the solution, and the resultingmixture was stirred at room temperature for 4 hours. To the reactionmixture, 1 mol/L aqueous sodium hydroxide (30 μL) was added, and themixture was stirred at room temperature for 2 hours. To the reactionmixture, 1 mol/L hydrochloric acid was added, the mixture was extractedwith ethyl acetate, and then the organic layer was dried. The solventwas evaporated under reduced pressure, and the resulting residue waspurified by column chromatography (chloroform/methanol) to obtain thetitle compound (0.7 mg).

(LCMS m/z 552.2 (MH⁺), retention time 1.60 minutes, LC conditions LC-1)

Example 13:5-(2-(4-(3-Hydroxy-4-(3-(thiazol-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 6by using 2-bromothiazole (16.5 mg, TCI) instead of4-bromothiophene-2-carbonitrile to obtain the title compound (0.4 mg).

(LCMS m/z 528.2 (MH⁺), retention time L43 minutes, LC conditions LC-1)

Example 14:5-(2-(4-(4-(3-((3-Cyanothiophen-2-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

Methyl5-(2-(4-(3-((tert-Butyldimethylsilyl)oxy)-4-(3-((3-cyanothiophen-2-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-14)

To a solution of the intermediate Z-22 (16 mg) in acetonitrile (1 mL),bis(acetonitrile)palladium chloride (0.7 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (4.0 mg), cesiumcarbonate (10.9 mg), and 2-bromothiophene-3-carbonitrile (15.7 mg,MAYBRIDGE) were successively added, and the resulting mixture wasstirred at 60° C. for 18 hours under a nitrogen gas atmosphere. Thereaction mixture was filtered through filter paper covered with Celite,the residue remained on Celite was washed with a mixed solvent ofchloroform and methanol (9:1). The filtrate and the wash liquid weremixed, the solvent was evaporated under reduced pressure, and then theresulting residue was purified by column chromatography (hexane/ethylacetate) to obtain the title compound (7.9 mg).

(Intermediate Z-7-14: LCMS m/z 680.5 (MH⁺), retention time 2.52 minutes,LC conditions LC-1)

[Step b]

5-(2-(4-(4-(3-((3-Cyanothiophen-2-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-14 (7.9 mg) in tetrahydrofuran (1 mL)was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solution intetrahydrofuran, 0.5 mL) was added to the solution, and the resultingmixture was stirred at room temperature for 18 hours. To the reactionmixture, methanol (0.5 mL), and 1 mol/L aqueous sodium hydroxide (0.5mL) were added, and the resulting mixture was stirred at roomtemperature for 2.5 hours. To the reaction mixture, 1 mol/L hydrochloricacid was added, the mixture was extracted with ethyl acetate, and theorganic layer was dried. The solvent was evaporated under reducedpressure, and the resulting residue was purified by columnchromatography (chloroform/methanol) to obtain the title compound (3.0mg).

(LCMS m/z 552.0 (MH⁺), retention time 1.57 minutes, LC conditions LC-1)

Example 15:5-(2-(4-(3-Hydroxy-4-(3-(phenylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 14by using bromobenzene (12.3 mg, TCI) instead of2-bromothiophene-2-carbonitrile to obtain the title compound (2.4 mg).

(LCMS m/z 521.0 (MH⁺), retention time 1.71 minutes, LC conditions LC-1)

Example 16:5-(2-(4-(3-Hydroxy-4-(3-((2-methoxyphenyl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 14by using 1-bromo-2-methoxybenzene (14.7 mg, WAKO) instead of2-bromothiophene-2-carbonitrile to obtain the title compound (1.6 mg).

(LCMS m/z 551.3 (MH⁺), retention time 1.64 minutes, LC conditions LC-1)

Reference Example A-10-2: (3-Chlorothiophen-2-yl)methanol (IntermediateA-10-2)

A solution of 3-chlorothiophene-2-carboxylic acid (4.47 g, ALDRICH) intetrahydrofuran (88.1 mL) was cooled to 0° C. under a nitrogen gasatmosphere, a 1 mol/L solution of borane-tetrahydrofuran complex intetrahydrofuran (49.7 mL) was added dropwise to the solution, and theresulting mixture was stirred at room temperature for 22 hours. Thereaction mixture was cooled to 0° C., methanol, water, and ethyl acetatewere added to the mixture, and the resulting mixture was stirred. Theorganic solvent was evaporated under reduced pressure, then ethylacetate was added to the residue, and the organic layer was successivelywashed with saturated aqueous sodium hydrogencarbonate, and saturatedbrine, and dried. The solvent was evaporated under reduced pressure toobtain the title compound (4.62 g).

(Intermediate A-10-2: Rf (TLC)=0.40 (hexane ethyl acetate=2:1))

Reference Example A-11-2: 3-Chloro-2-(bromomethyl)thiophene(Intermediate A-11-2)

A solution of the intermediate A-10-2 (4.62 g) in dichloromethane (110mL) was cooled to 0° C., triphenylphosphine (10.8 g), and carbontetrabromide (10.9 g) were added to the solution, and the resultingmixture was stirred at room temperature for 1 hour. To the reactionmixture, water, saturated brine, and ethyl acetate were added, themixture was stirred, and then the organic solvent was evaporated underreduced pressure. Ethyl acetate was added to the residue, and theorganic layer was successively washed with saturated brine, and dried.The organic solvent was evaporated under reduced pressure, and then amixed solvent of hexane and ethyl acetate (9:1) was added to theresulting residue to prepare a suspension, and the suspension wasfiltered through filter paper covered with silica gel. The solvent ofthe filtrate was evaporated under reduced pressure to obtain the titlecompound (9.09 g).

(Intermediate A-11-2: Rf (TLC)=0.56 (hexane:ethyl acetate=8:1))

Reference Example A-12-2: 2-(3-Chlorothiophen-2-yl)acetonitrile(Intermediate A-12-2)

To the intermediate A-11-2 (9.09 g), dimethyl sulfoxide (42 mL), andacetonitrile (126 mL) were added, the mixture was cooled to 0° C., thensodium cyanide (2.46 g) was added to the mixture, and the resultingmixture was stirred at room temperature for 2 hours. To the reactionmixture, water, saturated brine, and ethyl acetate were added, theresulting mixture was stirred, and then the organic layer was washedwith saturated brine, and dried. The solvent was evaporated underreduced pressure, and then the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (3.41g).

(Intermediate A-12-2: Rf (TLC)=0.20 (hexane:ethyl acetate=8:1))

Reference Example A-13-2: Ethyl 2-(3-chlorothiophen-2-yl)acetate(Intermediate A-13-2)

To a solution of the intermediate A-12-2 (3.41 g) in ethanol (36 mL),water (0.46 mL) was added, the mixture was cooled to 0° C., and thenconcentrated sulfuric acid (6.3 mL) was added portionwise to themixture. The reaction mixture was stirred at 85° C. for 88 hours, andthen cooled to 0° C., and saturated aqueous sodium hydrogencarbonate wasadded to the mixture until the mixture became neutral. Ethyl acetate wasadded to the mixture, and the resulting mixture was stirred, and thenconcentrated under reduced pressure. Ethyl acetate was added to themixture, and the organic layer was successively washed with saturatedaqueous sodium hydrogencarbonate, and saturated brine, and dried. Thesolvent was evaporated under reduced pressure to obtain the titlecompound (4.56 g).

(Intermediate A-13-2: Rf (TLC)=0.31 (hexane:ethyl acetate=8:1))

Reference Example A-14-2: 2-(3-Chlorothiophen-2-yl)ethanol (IntermediateA-14-2)

A solution of the intermediate A-13-2 (4.56 g) in tetrahydrofuran (108mL) was cooled to 0° C. under a nitrogen gas atmosphere, lithiumaluminum hydride (1.48 g) was added to the solution, and the mixture wasstirred for 0.7 hour. To the reaction mixture, water, diethyl ether, and1 mol/L hydrochloric acid were added, the resulting mixture was stirred,and then the organic layer was successively washed with 1 mol/Lhydrochloric acid, saturated aqueous sodium hydrogencarbonate, andsaturated brine, and dried. The solvent was evaporated under reducedpressure to obtain the title compound (3.67 g).

(Intermediate A-14-2: Rf (TLC)=0.13 (hexane:ethyl acetate=4:1))

Reference Example A-2-3: (2-(3-Chlorothiophen-2-yl)ethoxy)(tert-butyl)dimethylsilane (Intermediate A-2-3)

The intermediate A-2-3 was synthesized according to the method describedin Reference Example A-2 by using the intermediate A-14-2 (3.67 g)instead of 2-(thiophen-2-yl)ethanol, and thus the title compound (4.29g) was obtained.

(Intermediate A-2-3: Rf (TLC)=0.61 (hexane:ethyl acetate=4:1))

Reference Example A-3-3:4-Chloro-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylicAcid (Intermediate A-3-3)

The intermediate A-3-3 was synthesized according to the method describedin Reference Example A-3 by using the intermediate A-2-3 (3.03 g)instead of the intermediate A-2, and thus the title compound (3.41 g)was obtained.

(Intermediate A-3-3: Rf (TLC)=0.11 (hexane:ethyl acetate=4:1))

Reference Example A-4-3: Methyl4-chloro-5-(2-hydroxyethyl)thiophene-2-carboxylate (Intermediate A-4-3)

The intermediate A-4-3 was synthesized according to the method describedin Reference Example A-4 by using the intermediate A-3-3 (4.35 g)instead of the intermediate A-3, and thus the title compound (2.23 g)was obtained.

(Intermediate A-4-3: Rf (TLC)=0.38 (hexane:ethyl acetate=1:1))

Reference Example A-5-3: Methyl4-chloro-5-(2-bromoethyl)thiophene-2-carboxylate (Intermediate A-5-3)

The intermediate A-5-3 was synthesized according to the method describedin Reference Example A-5 by using the intermediate A-4-3 (2.23 g)instead of the intermediate A-4, and thus the title compound (3.18 g)was obtained.

(Intermediate A-5-3: Rf (TLC)=0.52 (hexane:ethyl acetate=2:1))

Reference Example A-6-3: tert-Butyl2-(2-(3-chloro-5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate(Intermediate A-6-3)

The intermediate A-6-3 was synthesized according to the method describedin Reference Example A-6 by using the intermediate A-5-3 (3.18 g)instead of the intermediate A-5, and thus the title compound (3.29 g)was obtained.

(Intermediate A-5-3: Rf (TLC)=0.24 (hexane:ethyl acetate=2:1))

Reference Example Z-1-3: tert-Butyl 2(2-(3-chloro-5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-(((2-chloroethyl)thio)carbonyl)hydrazinecarboxylate(Intermediate Z-1-3)

The intermediate Z-1-3 was synthesized according to the method describedin Reference Example Z-1-2 by using the intermediate A-6-3 (1.79 g)instead of the intermediate A-6-2, and thus the title compound (2.36 g)was obtained.

(Intermediate Z-1-3: Rf (TLC)=0.24 (hexane:ethyl acetate=4:1))

Reference Example Z-2-3: Methyl4-chloro-5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate(Intermediate Z-2-3)

The intermediate Z-2-3 was synthesized according to the method describedin Reference Example Z-2-2 by using the intermediate Z-1-3 (2.36 g)instead of the intermediate Z-1-2, and thus the title compound (1.73 g)was obtained.

(Intermediate Z-2-3: Rf (TLC)=0.69 (hexane:ethyl acetate=1:1))

Reference Example Z-3-3: Methyl4-chloro-5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-3-3)

The intermediate Z-3-3 was synthesized according to the method describedin Reference Example Z-3-2 by using of the intermediate Z-2-3 (1.73 g)instead of the intermediate Z-2-2, and thus the title compound (1.04 g).

(Intermediate Z-3-3: Rf (TLC)=0.23 (hexane:ethyl acetate=1:1))

Reference Example Z-4-3: Methyl4-chloro-5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4-3)

The intermediate Z-4-3 was synthesized according to the method describedin Reference Example Z-4 by using the intermediate Z-3-3 (0.30 g)instead of the intermediate Z-3, and the intermediate C-3-2 (the wholeamount of the intermediate C-3-2 produced and obtained according to themethod described in Reference Example C-3-2 using 2.56 g of the startingmaterial, intermediate C-2-2) instead of the intermediate C-3, and thusthe title compound (0.64 g) was obtained.

(Intermediate Z-4-3: Rf (TLC)=0.31 (hexane:ethyl acetate=1:1))

Reference Example Z-14-4: Methyl4-chloro-5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-14-4)

To the intermediate Z-4-3 (429 mg), diethylamine (3.62 mL),3-ethynylthiophene (93 μL), copper(I) iodide (13.8 mg), andtetrakis(triphenyl-phosphine)palladium (41.8 mg) were successivelyadded, and the mixture was stirred at room temperature for 3.5 hoursunder a nitrogen gas atmosphere. To the reaction mixture, 1 mol/Lhydrochloric acid was added, and the mixture was extracted twice withethyl acetate. The organic layer was successively washed with saturatedbrine, saturated aqueous sodium hydrogencarbonate, and saturated brine,and dried. The solvent was evaporated under reduced pressure, and theresulting residue was purified by column chromatography(chloroform/methanol) to obtain the title compound (290.4 mg).

(Intermediate Z-14-4: LCMS m/z 573.2 (MH⁺), retention time 2.03 minutes,LC conditions LC-1)

Reference Example Z-17-17: Methyl4-chloro-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-17-17)

To a solution of the intermediate Z-14-4 (290 mg) in methanol (5 mL),tetrahydrofuran (1 mL) was added, the mixture was cooled to 0° C., andsodium borohydride (28.8 mg) was added portionwise to the mixture. Themixture was stirred at room temperature for 1.6 hours, then water wasadded to the mixture, and 1 N hydrochloric acid was further addedportionwise to the reaction mixture until the mixture became weaklyacidic. The mixture was extracted twice with ethyl acetate, and then theorganic layer was successively washed with saturated aqueous sodiumhydrogencarbonate, and saturated brine, and dried. The solvent wasevaporated under reduced pressure, and the resulting residue waspurified by column chromatography (chloroform/methanol) to obtain thetitle compound (183 mg).

(Intermediate Z-17-17: LCMS m/z 575.2 (MH⁺), retention time 1.98minutes, LC conditions LC-1)

Example 17:4-Chloro-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate Z-17-17 (183 mg) in tetrahydrofuran (2mL), methanol (2 mL) was added to the solution, the mixture was cooledto 0° C., and water (2.38 mL), and 4 mol/L aqueous lithium hydroxide(2.38 mL) were added to the mixture. The resulting mixture was stirredat room temperature for 1 hour, then water was added to the reactionmixture, and further 2 N hydrochloric acid was added portionwise to thereaction mixture until the mixture became weakly acidic. The reactionmixture was extracted twice with ethyl acetate, and then the organiclayer was successively washed with saturated aqueous sodiumhydrogencarbonate, and saturated brine, and dried. The solvent wasevaporated under reduced pressure, and the resulting residue waspurified by column chromatography (chloroform/methanol) to obtain thetitle compound (129 mg).

(LCMS m/z 561.25 (MH⁺), retention time 1.72 minutes, LC conditions LC-1)

Reference Example C-1: 2-(3-Bromo-4-methylphenyl)acetonitrile(Intermediate C-1)

To a solution of 2-bromo-1,4-dimethylbenzene (2 g, TCI) in carbontetrachloride (21.6 mL), N-bromosuccinimide (1.06 g), and benzoylperoxide (56.7 mg) were added, and the mixture was stirred at 85° C. for1.5 hours. To the reaction mixture, N-bromosuccinimide (1.06 g), andbenzoyl peroxide (56.7 mg) were added, and the resulting mixture wasfurther stirred at 85° C. for 4.5 hours. The reaction mixture was cooledto room temperature, and then filtered through filter paper, and theresidue remained on the filter paper was washed with dichloromethane.The filtrate and the wash liquid were mixed, and the solvent wasevaporated under reduced pressure. To the resulting residue, ethanol(10.8 mL), water (5.4 mL), and potassium cyanide (2.1 g) were added, andthe mixture was stirred at 100° C. for 5 hours. The reaction mixture wascooled to room temperature, and extracted with ethyl acetate, and thenthe organic layer was dried. The solvent was evaporated under reducedpressure, and the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (576mg).

(Intermediate C-1: Rf (TLC)=0.58 (hexane:ethyl acetate=2:1))

¹H-NMR (CDCl₃): δ (ppm) 7.51 (1H, s), 7.24 (1H, d, J=7.5 Hz), 7.18 (1H,d, J=7.5 Hz), 3.70 (2H, s), 2.40 (3H, s)

Reference Example C-2-4:2-(3-Bromo-4-methylphenyl)-N-methoxy-N-methylacetamide (IntermediateC-2-4)

To the intermediate C-1 (300 mg), water (7.1 mL) was added, the mixturewas cooled to 0° C., concentrated sulfuric acid (5.7 mL) was addedportionwise to the mixture, and then the resulting mixture was stirredat 105° C. for 15 hours. The reaction mixture was cooled to roomtemperature, and then ethyl acetate was added for extraction. Hexane wasadded to the aqueous layer for extraction, and then the aqueous layerwas further extracted with diethyl ether. The resulting organic layerswere mixed, and washed with saturated aqueous sodium hydrogencarbonate,and dried. The solvent was evaporated under reduced pressure, and to theresulting residue. N,N-dimethylformamide (14.3 mL),N,O-dimethylhydroxylamine hydrochloride (557 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (821 mg),N,N-dimethyl-4-aminopyridine (17 mg), and diisopropylethylamine (L2 mL)were successively added, and the resulting mixture was stirred at roomtemperature for 17 hours. To the reaction mixture, diethyl ether wasadded, and then the organic layer was washed 3 times with 1 mol/Lhydrochloric acid, and once with saturated brine, and dried. The solventwas evaporated under reduced pressure, and the resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (221 mg).

(Intermediate C-2-4: LCMS m/z 272.3 (MH⁺), retention time 1.57 minutes,LC conditions LC-1)

Reference Example Z-4-4: Methyl5-(2-(4-(4-(3-bromo-4-methylphenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4-4)

A solution of the intermediate C-2-4 (142.6 mg) in dimethoxyethane (2.85mL) was cooled to 0° C. under a nitrogen atmosphere. To the reactionmixture, vinylmagnesium bromide (1 mol/L solution in tetrahydrofuran,790 μL, ALDRICH) was added, and the mixture was stirred for 4 hours. Tothe reaction mixture, 2 mol/L hydrochloric acid was added, and themixture was stirred for 1 minute. Ethyl acetate was added to the mixturefor extraction, and the organic layer was dried. The solvent wasevaporated under reduced pressure, ethanol (3 mL), water (3 mL), and theintermediate Z-3 (100 mg) were added to the resulting residue, and themixture was stirred overnight at 110° C. To the reaction mixture,saturated brine was added, and the mixture was extracted withchloroform. The organic layer was dried, and the solvent was evaporatedunder reduced pressure. The resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound(151.9 mg).

(Intermediate Z-4-4: LCMS m/z 525.1 (MH⁺), retention time 1.87 minutes,LC conditions LC-1)

Reference Example Z-6-4: Methyl5-(2-(4-(4-(3-bromo-4-methylphenyl)-3-((tert-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-6-4)

To a solution of the intermediate Z-4-4 (152 mg) in methanol (2.9 mL),tetrahydrofuran (5 mL) was added, the mixture was cooled to 0° C., andsodium borohydride (16.4 mg) was added to the mixture. The mixture wasstirred at 0° C. for 1 hour, and then diluted hydrochloric acid wasadded portionwise to the reaction mixture until the mixture becameweakly acidic. The organic solvent was evaporated under reducedpressure, then ethyl acetate was added to the residue for extraction,the organic layer was dried, and then the solvent was evaporated underreduced pressure. To the resulting residue, N,N-dimethylformamide (1.4mL), imidazole (98 mg), and tert-butyldimethylchlorosilane (131 mg) weresuccessively added, and the resulting mixture was stirred overnight atroom temperature. To the reaction mixture, 2 mol/L hydrochloric acid wasadded, and the mixture was extracted with ethyl acetate. The organiclayer was successively washed with water, and saturated brine, and thendried. The solvent was evaporated under reduced pressure, and theresulting residue was purified by column chromatography (hexane/ethylacetate) to obtain the title compound (165.7 mg).

(Intermediate Z-6-4: LCMS m/z 641.2 (MH⁺), retention time 2.02 minutes,LC conditions LC-6)

Reference Example Z-7-18: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(4-methyl-3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-18)

The intermediate Z-7-18 was synthesized according to the methoddescribed in Reference Example C-4 by using the intermediate Z-6-4 (20.0mg) instead of the intermediate C-2-2, and thus the title compound (23.4mg) was obtained.

(Intermediate Z-7-18: LCMS m/z 669.3 (MH⁺), retention time 2.25 minutes,LC conditions LC-6)

Example 18:5-(2-(4-(3-Hydroxy-4-(4-methyl-3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-18 (23.4 mg) in tetrahydrofuran (0.93mL) was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solutionin tetrahydrofuran, 93 μL) was added to the solution, and the mixturewas stirred at room temperature for 1.5 hours. To the reaction mixture,tetrabutylammonium fluoride (1 mol/L solution in tetrahydrofuran, 93 μL)was added, and the mixture was stirred at room temperature for further1.5 hours. To the reaction mixture, methanol (0.93 mL), and 1 mol/Laqueous sodium hydroxide (0.93 mL) were added, and the mixture wasstirred overnight at room temperature. To the reaction mixture, 1 mol/Lhydrochloric acid was added, the mixture was extracted with ethylacetate, and the organic layer was dried. The solvent was evaporatedunder reduced pressure, and the resulting residue was purified by columnchromatography (chloroform/methanol) to obtain the title compound (17.3mg).

(LCMS m/z 541.2 (MH⁺), retention time 1.74 minutes, LC conditions LC-1)

Reference Example C-1-2: 2-(3-Bromo-5-methylphenyl)acetonitrile(Intermediate C-1-2)

The intermediate C-1-2 was synthesized according to the method describedin Reference Example C-1 by using 1-bromo-3,5-dimethylbenzene (4.00 g,TCI) instead of 2-bromo-1,4-dimethylbenzene, and thus the title compound(2.34 g) was obtained.

(Intermediate C-1-2: Rf (TLC)=0.64 (hexane:ethyl acetate=2:1))

¹H-NMR (CDCl₃): δ (ppm) 7.31 (1H, m), 7.28 (1H, m), 7.09 (1H, m), 3.69(2H, s), 2.35 (3H, s)

Reference Example C-2-5:2-(3-Bromo-5-methylphenyl)-N-methoxy-N-methylacetamide (IntermediateC-2-5)

The intermediate C-2-5 was synthesized according to the method describedin Reference Example C-2-4 by using the intermediate C-1-2 (500 mg)instead of the intermediate C-1, and thus the title compound (553 mg)was obtained.

(Intermediate C-2-5: LCMS m/z 272.3 (MH⁺), retention time 1.57 minutes,LC conditions LC-1)

Reference Example Z-4-5: Methyl5-(2-(4-(4-(3-bromo-5-methylphenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4-5)

The intermediate Z-4-5 was synthesized according to the method describedin Reference Example Z-4-4 by using the intermediate C-2-5 (142.6 mg)instead of the intermediate C-2-4, and thus the title compound (149.9mg) was obtained.

(Intermediate Z-4-5: LCMS m/z 525.1 (MH⁺), retention time 1.88 minutes,LC conditions LC-1)

Reference Example Z-6-5: Methyl5-(2-(4-(4-(3-bromo-5-methylphenyl)-3-((tert-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-6-5)

The intermediate Z-6-5 was synthesized according to the method describedin Reference Example Z-6-4 by using the intermediate Z-4-5 (149.9 mg)instead of the intermediate Z-4-4, and thus the title compound (163.0mg) was obtained.

(Intermediate Z-6-5: LCMS m/z 641.3 (MH⁺), retention time 2.00 minutes,LC conditions LC-6)

Reference Example Z-7-19: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-methyl-5-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-19)

The intermediate Z-7-19 was synthesized according to the methoddescribed in Reference Example C-4 by using the intermediate Z-6-5 (20mg) instead of the intermediate C-2-2, and thus the title compound (19.2mg) was obtained.

(Intermediate Z-7-19: LCMS m/z 669.3 (MH⁺), retention time 2.25 minutes,LC conditions LC-6)

Example 19:5-(2-(4-(3-Hydroxy-4-(3-methyl-5-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 18by using the intermediate Z-7-19 (19.2 mg) instead of the intermediateZ-7-18 to obtain the title compound (13.5 mg).

(LCMS m/z 541.2 (MH⁺), retention time 1.74 minutes, LC conditions LC-1)

Reference Example C-2-6:2-(3-Bromo-4-fluorophenyl)-N-methoxy-N-methylacetamide (IntermediateC-2-6)

To a solution of 2-(3-bromo-4-fluorophenyl)acetic acid (500 mg) indichloromethane (43 mL), N,O-dimethylhydroxylamine hydrochloride (419mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (494mg), N,N-dimethyl-4-aminopyridine (26 mg), and diisopropylethylamine(1.2 mL) were successively added, and the resulting mixture was stirredovernight at room temperature. The solvent of the reaction mixture wasevaporated under reduced pressure, ethyl acetate was added to theresidue, and then the organic layer was successively washed with 1 mol/Lhydrochloric acid, and saturated brine, and dried. The solvent wasevaporated under reduced pressure, and the resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (449 mg).

(Intermediate C-2-6: LCMS m/z 276.2 (MH⁺), retention time 1.37 minutes,LC conditions LC-1)

Reference Example Z-4-6: Methyl5-(2-(4-(4-(3-bromo-4-fluorophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-4-6)

The intermediate Z-4-6 was synthesized according to the method describedin Reference Example Z-4-4 by using the intermediate C-2-6 (450.5 mg)instead of the intermediate C-2-4, and thus the title compound (562.8mg) was obtained.

(Intermediate Z-4-6: LCMS m/z 529.1 (MH⁺), retention time 1.78 minutes,LC conditions LC-1)

Reference Example Z-6-6: Methyl5-(2-(4-(4-(3-bromo-4-fluorophenyl)-3-((tert-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-6-6)

The intermediate Z-6-6 was synthesized according to the method describedin Reference Example Z-6-4 by using the intermediate Z-4-6 (562.8 mg)instead of the intermediate Z-4-4, and thus the title compound (719.7mg) was obtained.

(Intermediate Z-6-6: LCMS m/z 645.3 (MH⁺), retention time 1.64 minutes,LC conditions LC-6)

Reference Example Z-7-20: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(4-fluoro-3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-20)

The intermediate Z-7-20 was synthesized according to the methoddescribed in Reference Example C-4 by using the intermediate Z-6-6 (15.0mg) instead of the intermediate C-2-2, and thus the title compound (15.3mg) was obtained.

(Intermediate Z-7-20: LCMS m/z 673.4 (MH⁺), retention time 1.87 minutes,LC conditions LC-6)

Example 20:5-(2-(4-(4-(4-Fluoro-3-(thiophen-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

A solution of the intermediate Z-7-20 (15.3 mg) in tetrahydrofuran (345μL) was cooled to 0° C., tetrabutylammonium fluoride (1 mol/L solutionin tetrahydrofuran, 69 μL) was added to the solution, and the mixturewas stirred at room temperature for 4 hours. To the reaction mixture,tetrabutylammonium fluoride (1 mol/L solution in tetrahydrofuran, 70 andtetrahydrofuran (350 μL) were added, and the mixture was stirred at roomtemperature for further 2.5 hours. To the reaction mixture, methanol(345 μL), and 1 mol/L aqueous sodium hydroxide (345 μL) were added, andthe mixture was stirred overnight at room temperature. To the reactionmixture, 1 mol/L hydrochloric acid, and ethyl acetate were added forextraction, and then the organic layer was washed with 1 mol/Lhydrochloric acid, and dried. The solvent was evaporated under reducedpressure, and the resulting residue was purified with an anion exchangeresin to obtain the title compound (15.3 mg).

(LCMS m/z 545.2 (MH⁺), retention time 1.67 minutes, LC conditions LC-1)

Reference Example X-1: 4-Phenylthiophene-3-carboaldehyde (IntermediateX-1)

To a solution of (4-formylthiophen-3-yl)boronic acid (0.5 g,COMBI-BLOCKS) in n-butanol (32 mL), bromothiophene (1.0 mL, TCI), water(6.4 mL), palladium acetate (36 mg),2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (132 mg), and potassiumphosphate (1.36 g) were successively added, and the resulting mixturewas stirred overnight at 95° C. under a nitrogen gas atmosphere. To thereaction mixture, diethyl ether was added, and then the organic layerwas washed with water, and dried. The solvent was evaporated underreduced pressure, and the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (445mg) was obtained.

(Intermediate X-1: LCMS m/z 189.0 (MH⁺), retention time 1.54 minutes, LCconditions LC-1)

Reference Example X-2: 3-Ethynyl-4-phenylthiophene (Intermediate X-2)

To a solution of dimethyl (1-diazo-2-oxopropyl)phosphonate (727 mg, TCI)in methanol (14.9 mL), the intermediate X-1 (445 mg) was added, and themixture was cooled to 0° C. To the reaction mixture, potassium carbonate(686 mg) was added portionwise, and the mixture was stirred overnight atroom temperature. To the reaction mixture, saturated aqueous ammoniumchloride was added, and the mixture was extracted with diethyl ether.The organic layer was dried, then the solvent was evaporated underreduced pressure, and the resulting residue was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (384mg).

(Intermediate X-2: LCMS m/z 185.1 (MH⁺), retention time 1.81 minutes, LCconditions LC-1)

Reference Example Z-7-21: Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((4-phenylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-7-21)

The intermediate Z-7-21 was synthesized according to the methoddescribed in Reference Example C-4 by using the intermediate Z-6 (15.0mg) instead of the intermediate C-2-2, and the intermediate X-2 (9.4 mg)instead of 3-ethynylthiophene, and thus the title compound (15.2 mg) wasobtained.

(Intermediate Z-7-21: LCMS m/z 731.21 (MH⁺), retention time 2.41minutes, LC conditions LC-6)

Example 21:5-(2-(4-(3-Hydroxy-4-(3-((4-phenylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 18by using the intermediate Z-7-21 (15.2 mg) instead of the intermediateZ-7-18 to obtain the title compound (5.7 mg).

(LCMS m/z 603.0 (MH⁺), retention time 1.87 minutes, LC conditions LC-1)

Reference Example T-2:2-(4-Bromothiophen-2-yl)-N-methoxy-N-methylacetamide (Intermediate T-2)

A solution of 2-(4-bromothiophen-2-yl)acetic acid (1.0 g) indichloromethane (9 mL) was cooled to 0° C., N,O-dimethylhydroxylaminehydrochloride (882 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (1.04 g), N,N-dimethyl-4-aminopyridine (55 mg), anddiisopropylethylamine (3.38 mL) were added to the solution, thendiisopropylethylamine (0.35 mL) was further added, and the mixture wasstirred at room temperature for 41 hours. The reaction mixture wasconcentrated, and then ethyl acetate and water were added, 1 mol/Lhydrochloric acid was added for partitioning, and extraction was furtherperformed twice with ethyl acetate. The organic layer was successivelywashed with 1 mol/L hydrochloric acid, water, saturated aqueous sodiumhydrogencarbonate, water, and saturated brine, dried over magnesiumsulfate, and concentrated under reduced pressure to obtain the titlecompound (891 mg).

(Intermediate T-2: LCMS m/z 264.2, 266 (MH⁺), retention time 1.36minutes, LC conditions LC-1)

Reference Example T-3:1-(4-Bromothiophen-2-yl)-4-(methoxy(methyl)amino)butan-2-one(Intermediate T-3)

A solution of the intermediate T-2 (200 mg) in 1,2-dimethoxyethane (7mL) was cooled to 0° C. under a nitrogen atmosphere. To the reactionmixture, vinylmagnesium bromide (1 mol/L solution in tetrahydrofuran,1.1 mL, ALDRICH) was added, and the mixture was stirred at the sametemperature for 2 hours and 50 minutes. To the reaction mixture, 1 mol/Lhydrochloric acid was added to make the mixture acidic still at 0° C.,and then the mixture was extracted 3 times with ethyl acetate. Theorganic layer was washed with water, and saturated brine, dried overmagnesium sulfate, and then concentrated under reduced pressure toobtain the title compound (172.8 mg).

(Intermediate T-3: LCMS m/z 292.1, 294 (MH⁺), retention time 1.55minutes, LC conditions LC-1)

Reference Example T-4: Methyl5-(2-(4-(4-(4-bromothiophen-2-yl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate T-4)

The intermediate T-3 (172.8 mg), and the intermediate Z-3 (181 mg) weredissolved in ethanol (5 mL), water (5 mL) was added to the solution, andthe mixture was stirred at 105° C. for 16.5 hours. The reaction mixturewas poured into saturated brine diluted with water, and the mixture wasextracted 3 times with chloroform. The organic layer was washed withsaturated brine, and dried over magnesium sulfate, and then the solventwas evaporated under reduced pressure. The resulting residue waspurified by column chromatography (n-hexane/ethyl acetate) to obtain thetitle compound (112.0 mg).

(Intermediate T-4: LCMS m/z 517.0, 519.1 (MH⁺), retention time 1.76minutes, LC conditions LC-1)

Reference Example T-5: Methyl5-(2-(4-(4-(4-bromothiophen-2-yl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate T-5)

A solution of the intermediate T-4 (112 mg) in methanol (2 mL) wascooled to 0° C., sodium borohydride (12.3 mg) was added portionwise tothe solution. The reaction mixture was stirred at room temperature for 3hours, and then poured into water, and the mixture was extracted 3 timeswith ethyl acetate. The organic layer was washed with water, and thenwith saturated brine, and dried over magnesium sulfate, and then thesolvent was evaporated under reduced pressure to obtain the titlecompound (99.6 mg).

(Intermediate T-5: LCMS m/z 519.08, 521.08 (MH⁺), retention time 1.70minutes, LC conditions LC-1)

Reference Example T-6: Methyl5-(2-(4-(3-acetoxy-4-(4-bromothiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate T-6)

A solution of the intermediate T-5 (95.7 mg) in dichloromethane (1.8 mL)was cooled to 0° C., acetic anhydride (348 μL), and pyridine (741 μL)were added to the solution, the mixture was stirred at room temperaturefor 17 hours, then acetic anhydride (348 μL) was added to the mixture,and the resulting mixture was stirred at room temperature for further 4hours. Water was added to the reaction mixture, and then the mixture wasextracted 3 times with chloroform. The organic layer was washed withsaturated aqueous sodium hydrogencarbonate, and then with saturatedbrine, and dried over magnesium sulfate, and then the solvent wasevaporated under reduced pressure. The resulting residue was purified bycolumn chromatography (n-hexane/ethyl acetate) to obtain the titlecompound.

(Intermediate T-6: LCMS m/z 561.1, 563.1 (MH⁺), retention time 1.89minutes, LC conditions LC-1)

Example 22:5-(2-(4-(3-Hydroxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

Methyl5-(2-(4-(3-acetoxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate T-7)

To a solution of the intermediate T-6 (17.5 mg) in acetonitrile (1 mL),bis(acetonitrile)palladium chloride (1.2 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (6.7 mg), cesiumcarbonate (13.2 mg), and 3-ethynylthiophene (5.6 μL) were successivelyadded, and the resulting mixture was stirred at 60° C. for 17 hoursunder an argon gas atmosphere. The reaction mixture was filtered throughfilter paper covered with Celite, and the residue remained on Celite waswashed with ethyl acetate. The filtrate and the wash liquid were mixed,the solvent was evaporated under reduced pressure, and then theresulting residue was purified by column chromatography (n-hexane/ethylacetate) to obtain a mixture (18.6 mg) of the intermediate T-6 and thetitle compound. To a solution of the resulting mixture in acetonitrile(2 mL), bis(acetonitrile)palladium chloride (1.2 mg),2-dicyclohexyl-phosphino-2′,4′,6′-triisopropylbiphenyl (6.7 mg), cesiumcarbonate (13.2 mg), and 3-ethynylthiophene (6.14 μL) were successivelyadded, and the resulting mixture was stirred at 60° C. for 19 hoursunder an argon gas atmosphere. The reaction mixture was filtered throughfilter paper covered with Celite, and the residue remained on Celite waswashed with ethyl acetate. The filtrate and the wash liquid were mixed,the solvent was evaporated under reduced pressure, and then theresulting residue was purified by column chromatography (n-hexane/ethylacetate) to obtain the title compound (14.2 mg).

(Intermediate T-7: LCMS m/z 589.1 (MH⁺), retention time 2.03 minutes, LCconditions LC-1)

[Step b]

5-(2-(4-(3-Hydroxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate T-7 (14.2 mg) in tetrahydrofuran (0.36mL), 1 mol/L aqueous lithium hydroxide (0.36 mL) was added, and themixture was stirred overnight at room temperature. The reaction mixturewas cooled to 0° C., and 1 mol/L hydrochloric acid (0.36 mL) was addedto the mixture. The mixture was diluted with water, and extracted 3times with chloroform, then the organic layer was washed with saturatedbrine, and dried over magnesium sulfate, and the solvent was evaporatedunder reduced pressure. The resulting residue was purified by columnchromatography (chloroform/methanol) to obtain the title compound (3.5mg).

(LCMS m/z 533.0 (MH⁺), retention time 1.64 minutes, LC conditions LC-1)

Example 23:(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

(S)-Methyl5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-17-S)

The intermediate Z-17 (592 mg) was subjected to separation by HPLC usinga chiral column (HPLC apparatus was preparative purification apparatusproduced by Japan Waters, chiral column CHIRALCEL AD-H (DaicelCorporation), eluent ethanol, flow rate 0.5 mL/minute, retention time14.91 minutes) to obtain the title compound (194.2 mg).

[Step b]

(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 1by using the intermediate Z-17-S (41.4 mg) instead of the intermediateZ-17 to obtain the title compound (31.8 mg).

(LCMS m/z 527.2 (MH⁺), retention time 1.68 minutes, LC conditions LC-1)

Reference Example U: (S)-Methyl5-(2-(4-(4-(3-bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate U)

The intermediate Z-5 (1 g) was subjected to separation by HPLC using achiral column (HPLC apparatus was preparative purification apparatusproduced by Japan Waters, chiral column CHIRALCEL OJ-H (DaicelCorporation), eluent methanol, flow rate 0.5 mL/minute, retention time20.72 minutes) to obtain the title compound (309 mg).

Reference Example V-1: (S)-Methyl5-(2-(4-(4-(3-bromophenyl)-3-((tert-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate V-1)

Synthesis was performed according to the method described in ReferenceExample Z-6 by using the intermediate U (299.3 mg) instead of theintermediate Z-5 to obtain the title compound (333.9 mg).

(Intermediate V-1: LCMS m/z 627.35, 629.35 (MH⁺), retention time L79minutes, LC conditions NLC-1)

Reference Example V-2: (S)-Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((trimethylsilyl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate V-2)

Synthesis was performed according to the method described in ReferenceExample Z-21 by using the intermediate V-1 (333.9 mg) instead of theintermediate Z-6 to obtain the title compound (91.3 mg).

(Intermediate V-2: LCMS m/z 645.49 (MH⁺), retention time 2.38 minutes,LC conditions NLC-6)

Reference Example V-3: (S)-Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-ethynylphenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate V-3)

Synthesis was performed according to the method described in ReferenceExample Z-22 by using the intermediate V-2 (91.3 mg) instead of theintermediate Z-21 to obtain the title compound (91.6 mg).

(Intermediate V-3: LCMS m/z 573.45 (MH⁺), retention time 1.41 minutes,LC conditions NLC-6)

Example 24:(S)-5-(2-(4-(4-(3-((2-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

(S)-Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((2-cyanothiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-24-1)

To a solution of the intermediate V-3 (50.6 mg) in acetonitrile (1 mL),bis(acetonitrile)palladium chloride (2.0 mg),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (11.2 mg), cesiumcarbonate (51.1 mg), and 3-bromothiophene-2-carbonitrile (73.8 mg) weresuccessively added, and the resulting mixture was stirred at 60° C. for0.75 hour under a nitrogen gas atmosphere. The reaction mixture wasfiltered through filter paper covered with Celite, and the residueremained on Celite was washed with a mixed solvent of chloroform andmethanol (9:1). The filtrate and the wash liquid were mixed, the solventwas evaporated under reduced pressure, and then the resulting residuewas purified by column chromatography (hexane/ethyl acetate) to obtainthe title compound (10.1 mg).

(Intermediate Z-24-1: LCMS m/z 680.44 (MH⁺), retention time 1.73minutes, LC conditions NLC-6)

[Step b]

(S)-Methyl5-(2-(4-(4-(3-((2-cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-24-2)

To a solution of the intermediate Z-24-1 (10.1 mg) in tetrahydrofuran(0.5 mL), tetrabutylammonium fluoride (1 mol/L solution intetrahydrofuran, 44.6 μL) was added, and the mixture was stirred at roomtemperature for 1 hour. The reaction mixture was purified by columnchromatography (hexane/ethyl acetate) to obtain the title compound (9.3mg).

(Intermediate Z-24-2: LCMS m/z 566.35 (MH⁺), retention time 1.77minutes, LC conditions NLC-6)

[Step c]

(S)-5-(2-(4-(4-(3-((2-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate Z-24-2 (9.3 mg) in tetrahydrofuran(400 μL), 1 mol/L aqueous sodium hydroxide (197 μL) was added, and themixture was stirred at room temperature for 60 hours. To the reactionmixture, 1 mol/L hydrochloric acid (400 μL) was added, the mixture wasextracted 3 times with chloroform, and then the organic layer was washedwith saturated brine, and dried. The solvent was evaporated underreduced pressure. The resulting residue was purified by columnchromatography (methanol/chloroform) to obtain the title compound (7.5mg).

(LCMS m/z 552.30.2 (MH⁺), retention time 1.23 minutes, LC conditionsNLC-1)

Example 25:(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

[Step a]

(S)-Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-25-1)

Synthesis was performed according to the method described in Example 24,Step a by using 4-bromothiazole (28.6 μL) instead of3-bromothiophene-2-carbonitrile to obtain the title compound (21.0 mg).

(Intermediate Z-25-1: LCMS m/z 656.43 (MH⁺), retention time 1.37minutes, LC conditions NLC-1)

[Step b]

(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate Z-25-1 (21.0 mg) in tetrahydrofuran (1mL), tetrabutylammonium fluoride (1 mol/L solution in tetrahydrofuran,92.6 μL) was added, and the mixture was stirred at room temperature for3 hours. The reaction mixture was purified by column chromatography(hexane/ethyl acetate). To a solution of the resulting intermediate(12.2 mg) in tetrahydrofuran (540 μL), and methanol (270 μL), 1 mol/Laqueous sodium hydroxide (270 μL) was added, and the mixture was stirredat room temperature for 16 hours. To the reaction mixture, 1 mol/Lhydrochloric acid was added, the mixture was extracted 5 times withethyl acetate, and then the organic layer was washed with saturatedbrine, and dried. The solvent was evaporated under reduced pressure toobtain the title compound (12.5 mg).

(LCMS m/z 528.29 (MH⁺), retention time 1.08 minutes, LC conditionsNLC-1)

Example 26:(S)-5-(2-(4-(4-(3-(Furan-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 25by using 3-bromofuran (31.2 mg) instead of 4-bromothiazole, and then theresultant was purified by column chromatography (methanol/chloroform) toobtain the title compound (5.1 mg).

(LCMS m/z 511.35 (MH⁺), retention time 1.23 minutes, LC conditionsNLC-1)

Reference Example Z-27: (E)-Methyl5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-(2-(thiophen-3-yl)vinyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-27)

To a solution of the intermediate Z-6 (50.0 mg) in 1,4-dioxane (637 μL),(E)-4,4,5,5-tetramethyl-2-(2-(thiophen-3-yl)vinyl)-1,3,2-dioxaborolane(22.6 mg, ALDRICH), bis(triphenylphosphine)palladium chloride (5.6 mg),sodium carbonate (21.1 mg), and water (199 μL) were successively added,and the resulting mixture was stirred at 85° C. for 11 hours under anitrogen gas atmosphere. To the reaction mixture, water was added, andthe mixture was extracted with ethyl acetate. The organic layer wasdried, then the solvent was evaporated under reduced pressure, and theresulting residue was purified by column chromatography (hexane/ethylacetate) to obtain the title compound (44.5 mg).

(Intermediate Z-27: LCMS m/z 657.3 (MH⁺), retention time 1.93 minutes,LC conditions LC-6)

Example 27:(E)-5-(2-(4-(3-Hydroxy-4-(3-(2-(thiophen-3-yl)vinyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 18by using the intermediate Z-27 (24.8 mg) instead of the intermediateZ-7-18 to obtain the title compound (17.8 mg).

(LCMS m/z 529.1 (MH⁺), retention time 1.63 minutes, LC conditions LC-1)

Reference Example Z-14-5: Methyl4-chloro-5-(2-(2-oxo-4-(3-oxo-4-(3-(pyridin-2-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-14-5)

To the intermediate Z-4-3 (100 mg), diethylamine (850 μL),2-ethynylpyridine (22.8 μL), copper(I) iodide (2.9 mg), andtetrakis(triphenylphosphine)palladium (3.9 mg) were successively added,and the mixture was stirred at room temperature for 15.5 hours under anitrogen gas atmosphere. The reaction mixture was diluted with ethylacetate, then the solvent was evaporated, and the resulting residue waspurified by column chromatography (toluene/acetonitrile) to obtain thetitle compound (47.6 mg).

(Intermediate Z-14-5: LCMS m/z 570.425 (MH⁺), retention time 4.86minutes, LC conditions FLC-1))

Example 28:4-Chloro-5-(2-(4-(3-hydroxy-4-(3-(pyridin-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To a solution of the intermediate Z-14-5 (47.6 mg) in methanol (840 μL),sodium borohydride (4.8 mg) was added portionwise. The mixture wasstirred at room temperature for 0.5 hour, and then water, and ethylacetate were added to the mixture, and the organic layer wassuccessively washed with saturated aqueous sodium hydrogencarbonate, andsaturated brine, and dried. The solvent was evaporated under reducedpressure, and then tetrahydrofuran (640 μL), methanol (640 μL), and 2mol/L aqueous sodium hydroxide (640 μL) were added to the residue. Thereaction mixture was stirred at room temperature for 1.8 hours, and thencooled to 0° C., and 2 mol/L hydrochloric acid (640 μL) was addedportionwise to the mixture. To the reaction mixture, ethyl acetate wasadded, and the organic layer was washed with saturated brine, and dried.The solvent was evaporated under reduced pressure, and the resultingresidue was purified by thin layer chromatography(toluene/ethanol/acetic acid) to obtain the title compound (15.0 mg).

(LCMS m/z 556.023 (MH⁺), retention time 4.49 minutes, LC conditionsFLC-1)

Reference Example Z-29-1: 2-tert-Butyl 1-(2-chloroethyl)1-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazine-1,2-dicarboxylate(Intermediate Z-29-1)

To a solution of the intermediate A-6 (5.0 g) in acetonitrile (165 mL),potassium carbonate (0.46 g) was added, and the mixture was cooled to 0°C. 2-Chloroethyl chloroformate (2.07 mL) was slowly added to themixture, and the resulting mixture was stirred at the same temperaturefor 1 hour. To the reaction mixture, water was added, and the resultingmixture was warmed to room temperature, and extracted 3 times with ethylacetate. The organic layer was washed with water, and then withsaturated brine, and dried, and then the solvent was evaporated underreduced pressure to obtain the title compound (8.34 g).

(Intermediate Z-29-1: LCMS m/z 307.14, 309.11 (MH⁺-Boc), retention time1.64 minutes, LC conditions LC-1)

Reference Example Z-29-2: tert-Butyl3-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-oxo-1,3,4-oxadiazinane-4-carboxylate(Intermediate Z-29-2)

A solution of the intermediate Z-29-1 (8.34 g) in DMF (140 mL) wascooled to 0° C., sodium hydride (55%, 0.87 g) was added portionwise over30 minutes, and the mixture was stirred for 2 hours. The reactionmixture was returned to room temperature, and stirred for 1 hour, thensodium hydride (55%, 0.1 g) was added to the mixture again at 0° C., andthe mixture was stirred at room temperature for 15 hours. The reactionmixture was cooled to 0° C., water was added, and the resulting mixturewas extracted 4 times with ethyl acetate. The organic layer was washedtwice with water, and once with saturated brine, and dried, and then thesolvent was evaporated under reduced pressure. The resulting residue waspurified by column chromatography (hexane/ethyl acetate) to obtain thetitle compound (1.75 g).

(Intermediate Z-29-2: LCMS m/z 371.3 (MH⁺), retention time 1.51 minutes,LC conditions LC-1)

Reference Example Z-29-3: Methyl5-(2-(2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-29-3)

A solution of the intermediate Z-29-2 (1.75 g) in dichloromethane (25mL) was cooled to 0° C., trifluoroacetic acid (12.5 mL) was added to thesolution, and the mixture was stirred at room temperature for 1 hour.The reaction mixture was cooled to 0° C., neutralized with 5 M aqueoussodium hydroxide, and then extracted 3 times with chloroform. Theorganic layer was washed with water, and then dried, and the solvent wasevaporated under reduced pressure. The residue was purified with asilica gel short column (chloroform, then ethyl acetate), and then thedeposited solid was washed with diethyl ether to obtain the titlecompound (0.90 g).

(Intermediate Z-29-3: LCMS m/z 271.2 (MH⁺), retention time 0.94 minutes,LC conditions LC-1)

Reference Example Z-29-4: Methyl5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-29-4)

A solution of the intermediate C-2-2 (250 mg) in DME (5 mL) was cooledto 0° C. under a nitrogen flow, vinylmagnesium bromide (1 M solution,1.2 mL) was added dropwise to the solution, and the mixture was stirredat the same temperature for 2 hours. The reaction mixture was madeacidic by addition of 2 M hydrochloric acid, and then water was added tothe mixture, and the resulting mixture was extracted 3 times with ethylacetate. The organic layer was washed twice with water, and dried, andthen the solvent was evaporated under reduced pressure.

To a flask containing the intermediate Z-29-3 (110.7 mg), and water (3.3mL), a solution of the residue obtained above in ethanol (3.3 mL) wasadded, and the mixture was stirred at an external temperature of 110° C.for 15 hours. The reaction mixture was returned to room temperature, andthen poured into water, and the resulting mixture was extracted 3 timeswith ethyl acetate. The organic layer was washed with saturated brine,and dried, and then the solvent was evaporated under reduced pressure.The resulting residue was purified by column chromatography(hexane/ethyl acetate) to obtain the title compound (126.2 mg).

(Intermediate Z-29-4: LCMS m/z 541.2 (MH⁺), retention time L66 minutes,LC conditions LC-1)

Reference Example Z-29-5: Methyl5-(2-(4-(3-hydroxy-4-(3-iodophenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-29-5)

To a solution of the intermediate Z-29-4 (58.1 mg) in methanol (1 mL),sodium borohydride (6.1 mg) was added, and the mixture was stirred atroom temperature for 1 hour. Water was added to the mixture, and theresulting mixture was extracted 3 times with ethyl acetate. The organiclayer was washed with saturated brine, and dried, and then the solventwas evaporated under reduced pressure to obtain the title compound (61.4mg).

(Intermediate Z-29-5: LCMS m/z 545.2 (MH⁺), retention time 1.60 minutes,LC conditions LC-1)

Reference Example Z-29-6: Methyl5-(2-(4-(3-acetoxy-4-(3-iodophenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-29-6)

The intermediate Z-29-5 (61.4 mg) was dissolved in dichloromethane (2mL), acetic anhydride (0.27 mL), and pyridine (0.23 mL) were added tothe solution, and the mixture was stirred at room temperature 5.5 hours.To the reaction mixture, water was added, and the resulting mixture wasextracted 3 times with chloroform. The organic layer was washed withwater, and dried, and then the solvent was evaporated under reducedpressure. The residue was purified by short column chromatography toobtain the title compound (71.8 mg).

(Intermediate Z-29-6: LCMS m/z 587.26 (MH⁺), retention time 1.82minutes, LC conditions LC-1)

Reference Example Z-29-7: Methyl5-(2-(4-(3-acetoxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-29-7)

Synthesis was performed according to the method described in ReferenceExample Z-14 by using the intermediate Z-29-6 (71.8 mg) instead of theintermediate Z-4-2 to obtain the title compound (66.4 mg).

(Intermediate Z-29-7: LCMS m/z 567.36 (MH⁺), retention time 1.94minutes, LC conditions LC-1)

Example 29:5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

Synthesis was performed according to the method described in Example 22,Step b by using the intermediate Z-29-7 (66.4 mg) instead of theintermediate T-7 to obtain the title compound (45.6 mg).

(LCMS m/z 509.2 (MH⁺), retention time 1.53 minutes, LC conditions LC-1)

Reference Example D-2: Methyl 4-(3-bromophenyl)-3-oxobutanoate(Intermediate D-2)

To a solution of monomethyl potassium malonate (0.885 kg), THF (4.077kg), and magnesium chloride (0.47 kg) were added, and the mixture wasstirred at 50° C. for 10 minutes. To this mixture, a reaction mixtureobtained by adding a solution of carbonyldiimidazole (0.801 kg) in DMF(4.025 kg) to a solution of 3-bromophenylacetic acid (1.005 kg) in THF(2.023 kg), and stirring the mixture at room temperature for 1 hour wasadded. THF (0.508 kg) was further added to the reaction mixture, and theresulting mixture was stirred at 50° C. for 30 minutes. The reactionmixture was cooled to room temperature, ethyl acetate (8.071 kg) wasadded to the mixture, the organic layer was washed twice with 20%aqueous citric acid (6.032 kg), and then the solvent was evaporatedunder reduced pressure to obtain a concentrate (2.358 kg). To thisconcentrate, ethyl acetate (1.011 kg) was added to obtain a solutioncontaining the intermediate D-2 (3.369 kg). The solution containing theintermediate D-2 was mixed with a solution obtained by a similaroperation. To this mixture (6.770 kg), ethyl acetate (6.063 kg) wasadded, and 5% aqueous sodium hydrogencarbonate (10.055 kg), and sodiumchloride (0.506 kg) were further added to wash the organic layer. 5%Aqueous sodium hydrogencarbonate (10.054 kg), and sodium chloride (0.503kg) were added to further wash the organic layer. The organic layer wasfurther washed with 20% aqueous sodium chloride (10.064 kg), ethylacetate (1.01 kg) was added, and then the solvent was evaporated underreduced pressure to obtain the title compound (2.52 kg).

(LCMS m/z 268.9, 270.9 (MH⁻), retention time 1.44 minutes, LC conditionsNLC-1)

Reference Example D-3: Methyl (S)-4-(3-bromophenyl)-3-hydroxybutanoate(Intermediate D-3)

To the intermediate D-2 (2.52 kg), methanol (14.325 kg), water (0.237kg), and [NH₂Me₂][(RuCl((S)-dm-segphos))₂(u-Cl)₃] (82.46 g, TAKASAGO)were added, the mixture was stirred at 60° C. for 6 hours under ahydrogen atmosphere, and then the solvent was evaporated under reducedpressure. Toluene (20.70 kg), and QuadraSil AP (0.60 kg, JOHNSON) wereadded to the residue, the mixture was stirred at 60° C. for 1 hour, thenthe reaction mixture was filtered, and the filtration residue was washedwith toluene (2.066 kg). The solvent of the filtrate was evaporatedunder reduced pressure to obtain a concentrate (5.68 kg), and then, tothe concentrate, n-heptane (1.22 kg) was added dropwise at 10° C. over15 minutes, and n-heptane (1.22 kg) was further added dropwise at 5° C.over 50 minutes. The reaction mixture was stirred as it was at 5° C. for45 minutes, and then filtered. The filtration residue was washed with amixture of n-heptane (0.48 kg), and toluene (0.24 kg), and dried toobtain the title compound (1.923 kg).

(LCMS m/z 273.0, 275.1 (MH⁺), retention time 1.36 minutes, LC conditionsNLC-1) (Chiral LC: retention time 21.1 minutes, LC conditions ChiralLC-1)

Reference Example D-35: Methyl(S)-3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butanoate (IntermediateD-35)

To the intermediate D-3 (558 g), acetonitrile (1386 g), cesium carbonate(665.7 g), X-Phos (48.7 g, Nippon Kagaku), andbis(acetonitrile)palladium(II) dichloride (13.28 g, TCI) were added, anda solution of 3-ethynylthiophene (287.3 g) in acetonitrile (277.2 g) wasadded dropwise over 30 minutes under a nitrogen atmosphere. The reactionmixture was stirred at 40° C. for 30 minutes, and then at 60° C. for 90minutes. The reaction mixture was cooled to room temperature, toluene(1208.3 g) and water (3488.2 g) were added to the mixture, and theresulting mixture was stirred for 20 minutes, and then filtered throughCelite. The filtration residue was washed with toluene (2416.3 g), thefiltrate was stirred for 10 minutes, and then left standing until itseparated into an organic layer and an aqueous layer, and the aqueouslayer was removed. The organic layer was washed with water (2232.3 g),and then the solvent was evaporated under reduced pressure to obtain thetitle compound (753.9 g).

(LCMS m/z 301.2 (MH⁺), retention time 1.65 minutes, LC conditions NLC-1)

Reference Example D-50: Methyl(S)-3-((2-methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butanoate(Intermediate D-50)

To the intermediate D-35 (753.5 g), toluene (2588.1 g),diisopropylethylamine (334.3 g), and 2-methoxyethoxymethyl chloride(322.2 g) were added under a nitrogen atmosphere, and the mixture wasstirred at 80° C. for 150 minutes. The reaction mixture was cooled,water (3107.7 g) was added to the mixture, and the resulting mixture wasstirred for 10 minutes, and then filtered. The filtration residue waswashed with toluene (1031.6 g), the filtrate was left standing until itseparated into an organic layer and an aqueous layer, and the aqueouslayer was removed. The organic layer was washed with water (2071.9 g),and then the solvent was evaporated under reduced pressure to obtain anoily substance containing the intermediate D-50 (915.8 g). To this oilysubstance containing the intermediate D-50 (915.4 g), methanol (2310.7g), and activated carbon Shirasagi A (292.3 g, Japan EnviroChemicals)were added, and the mixture was stirred at room temperature for 1 hour,and then filtered with filter paper. The filtration residue was washedwith methanol (928.8 g), then the filtrate was filtered through amembrane filter having a pore diameter of 0.5 μm, and the filtrationresidue was washed with methanol (464.2 g). The filtrate wasconcentrated, toluene (2479.1 g), and QuadraSil MTU (349.6 g, JOHNSON)were added to the concentrate, and the mixture was stirred at 40° C. for1 hour, and then filtered with filter paper. The filtration residue waswashed with toluene (1008.0 g), then the filtrate was filtered through amembrane filter having a pore diameter of 0.5 μm, and the filtrationresidue was washed with toluene (503.8 g). The filtrate was concentratedunder reduced pressure to obtain the title compound (710.9 g).

(LCMS m/z 406.2 (M⁺NH₄ ⁺), retention time 1.88 minutes, LC conditionsNLC-1)

Reference Example D-60:(S)-3-((2-Methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butanal(Intermediate D-60)

To the intermediate D-50 (355.3 g), toluene (2740.1 g) was added under anitrogen atmosphere, and the mixture was cooled to −83° C. To thismixture, a 1 mol/L solution of diisobutylaluminum hydride in toluene(571.6 g) was added dropwise over 90 minutes. To the reaction mixture, 1mol/L hydrochloric acid (1884.1 g) was added, the mixture was stirred atroom temperature for 1 hour, toluene (64.3 g) was added to the mixture,the resulting mixture was stirred for 10 minutes, and then left standinguntil it separated into an organic layer and an aqueous layer, and theaqueous layer was removed. The organic layer was successively washedwith 1 mol/L hydrochloric acid (1004.9 g), 1% aqueous sodiumhydrogencarbonate (1997.8 g), and water (1978.1 g), and then filteredthrough a membrane filter having a pore diameter of 0.5 μm. Thefiltration residue was washed with toluene (214.3 g) to obtain a toluenesolution (4061.0 g) containing the title compound (248.5 g).

(LCMS m/z 376.2 (M+NH₄+), retention time 1.80 minutes, LC conditionsNLC-1)

Reference Example Z-70: Methyl(S)-5-(2-(4-(3-((2-methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-70)

To the intermediate Z-3 (286.3 g), toluene (1959.6 g), acetic acid(474.5 g), and sodium triacetoxyborohydride (498.5 g) were added under anitrogen atmosphere. The solvent of the solution in toluene (7927.0 g)containing the intermediate D-60 (460.28 g) was evaporated under reducedpressure to obtain a concentrated solution of the intermediate D-60(1503.9 g), and this solution was added to the reaction mixture at roomtemperature over 50 minutes. Toluene (245 g) was added to the reactionmixture, and the resulting mixture was stirred for 2 hours, and thensuccessively washed with 5% aqueous sodium hydrogencarbonate (7326.6 g),5% aqueous sodium hydrogencarbonate (7327.2 g), and water (7071.4 g).The solvent was evaporated under reduced pressure to obtain the titlecompound (903.8 g).

(LCMS m/z 629.29 (MH⁺), retention time 2.05 minutes, LC conditionsNLC-1)

Reference Example Z-17-S: Methyl(S)-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate(Intermediate Z-17-S)

To the intermediate Z-70 (903.5 g), methanol (4293.5 g), and activatedcarbon Shirasagi A (90.35 g, Japan EnviroChemicals) were added, thereaction mixture was stirred at 40° C. for 1 hour, and then filtered,and the filtration residue was washed with methanol (715.1 g). To thefiltrate, 36% hydrochloric acid (588.6 g) was added, the mixture wasstirred at 40° C. for 6 hours, then toluene (2406.8 g) was added to themixture, and the resulting mixture was cooled to room temperature. Thereaction mixture was successively washed with 5% aqueous sodiumhydrogencarbonate (9765.4 g), and water (1685.1 g), and then the solventwas evaporated under reduced pressure. The resulting residue waspurified by column chromatography (n-heptane/ethyl acetate) to obtainthe title compound (471.6 g).

(LCMS m/z 541.19 (MH⁺), retention time 1.87 minutes, LC conditionsNLC-1)

Example 30:(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylicAcid

To the intermediate Z-17-S (469.5 g), THF (2393.3 g), and activatedcarbon Shirasagi A (94.0 g, Japan EnviroChemicals) were added, thereaction mixture was stirred at room temperature for 1 hour, and thenfiltered, and the filtration residue was washed with THF (1597.1 g). Tothe filtrate, THF (378.9 g), methanol (1421.8 g), and 1 mol/L aqueoussodium hydroxide (1728.2 g) were added, and the mixture was stirred for17 hours. To the reaction mixture, toluene (2331.5 g), and water (1346.5g) were added, the mixture was stirred, and left standing until itseparated into an organic layer and an aqueous layer, and the organiclayer was removed. To the aqueous layer, a mixture of toluene (2332 g),THF (1596 g), and methanol (710 g) was added, the resulting mixture wasstirred, and left standing until it separated into an organic layer andan aqueous layer, and the organic layer was removed. Then, the washingwith a mixture of toluene, THF, and methanol was performed 3 times in asimilar manner. To the aqueous layer, toluene (4663.3 g) was added, themixture was stirred, and left standing until it separated into anorganic layer and an aqueous layer, and the organic layer was removed.To the aqueous layer, 1 mol/L hydrochloric acid was added until pH ofthe mixture became 7.0, then ethyl acetate (2425.9 g) was added, 1 mol/Lhydrochloric acid was further added until pH of the mixture became 2.2,the mixture was stirred for 10 minutes, and then left standing until itseparated into an organic layer and an aqueous layer, and the aqueouslayer was removed. To the organic layer, ethyl acetate (1347.1 g), andwater (897.5 g) were added, the mixture was stirred for 10 minutes, andthen left standing until it separated into an organic layer and anaqueous layer, and the aqueous layer was removed. The solvent of theorganic layer was evaporated under reduced pressure to obtain a residuecontaining the title compound (358.6 g). To this residue containing thetitle compound (358.3 g), methanol (1860.0 g), and activated carbonShirasagi A (47.6 g, Japan EnviroChemicals) were added, the mixture wasstirred at room temperature for 1 hour, and then filtered through amembrane filter having a pore diameter of 0.5 μm, and the filtrationresidue was washed with methanol (2231.2 g). To the filtrate, activatedcarbon Shirasagi A (188.6 g, Japan EnviroChemicals) was added, themixture was stirred at room temperature for 1 hour, and then filteredthrough filter paper, and the filtration residue was washed withmethanol (1490.5 g). The filtrate was filtered through a membrane filterhaving a pore diameter of 0.2 μm, and the filtration residue was washedwith methanol (743.7 g). To the filtrate, activated carbon Shirasagi A(94.0 g, Japan EnviroChemicals) was added, the mixture was stirred atroom temperature for 1 hour, and then filtered through a membrane filterhaving a pore diameter of 0.2 μm, and the filtration residue was washedwith methanol (2231.2 g). To the filtrate, activated carbon Shirasagi A(94.1 g, Japan EnviroChemicals) was added, the mixture was stirred atroom temperature for 1 hour, and then filtered through a membrane filterhaving a pore diameter of 0.2 μm, and the filtration residue was washedwith methanol (742.9 g). The solvent of the filtrate was evaporatedunder reduced pressure to obtain the title compound (160.1 g).

(LCMS m/z 527.2 (MH⁺), retention time 1.26 minutes, LC conditions NLC-1)(Chiral LC: retention time: 21.3 minutes, LC conditions Chiral LC-2)

The compound of Example 30 is the same as the compound of Example 23.

Comparative Example 1:4-(2-(4-(4-(3-Bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)benzoicAcid

The title compound can be obtained by the preparation method describedin International Patent Publication WO2006/080323 (Patent document 8),Example IAH-H072.

Preparation Example 1

Dichloromethane (20 mL) was added to poly(lactic-co-glycolic acid)(RESOMER RG504, produced by Evonik Industries, 2.0 g), which wasdissolved by using an ultrasonic washing machine, and the compound ofExample 23 (1.6 mg) was further added to the solution, and dissolved.This solution was added portionwise to a 0.1% aqueous solution ofpolyvinyl alcohol (300 mL) stirred at 3,000 rpm using a homomixer (MARKII produced by PRIMIX Corporation), and the mixture was stirred at roomtemperature for 10 minutes to obtain an o/w emulsion. This o/w emulsionwas stirred at room temperature for 16 hours, dichloromethane wasvolatilized to solidify the oil phase, and the resultant was centrifuged(3,000 rpm, 20° C., 15 minutes) by using a centrifugation machine. Afterthe supernatant was removed, the precipitates were dispersed in a 0.1%(w/v) solution of Tween 80, the dispersion was passed thorough sieves of53 μm and 20 μm, and the sample remained on the sieve of 20 μm wascentrifuged (3,000 rpm, 20° C., 15 minutes). The supernatant wasremoved, purified water was added to the precipitates, the mixture wascentrifuged again (3,000 rpm, 20° C., 15 minutes), and the supernatantwas removed. The precipitates were frozen at −80° C., and dried underreduced pressure (48 hours) to obtain drug-containing microspheres (1.2g) having an enclosed drug ratio of 0.06%.

Preparation Example 2

Dichloromethane (20 mL) was added to poly(lactic-co-glycolic acid)(RESOMER RG504, produced by Evonik Industries, 2.0 g), which wasdissolved by using an ultrasonic washing machine, and the compound ofExample 23 (20 mg) was further added to the solution, and dissolved.This solution was added portionwise to a 0.1% aqueous solution ofpolyvinyl alcohol (300 mL) stirred at 3,000 rpm using a homomixer (MARKII produced by PRIMIX Corporation), and the mixture was stirred at roomtemperature for 10 minutes to obtain an o/w emulsion. This o/w emulsionwas stirred at room temperature for 16 hours, dichloromethane wasvolatilized to solidify the oil phase, and the resultant was centrifuged(3,000 rpm, 20° C., 15 minutes) by using a centrifugation machine. Afterthe supernatant was removed, the precipitates were dispersed in a 0.1%(w/v) solution of Tween 80, the dispersion was passed thorough sieves of53 μm and 20 μm, and the sample remained on the sieve of 20 μm wascentrifuged (3,000 rpm, 20° C., 15 minutes). The supernatant wasremoved, then purified water was added to the precipitates, the mixturewas centrifuged again (3,000 rpm, 20° C., 15 minutes), and thesupernatant was removed. The precipitates were frozen at −80° C., anddried under reduced pressure (48 hours) to obtain drug-containingmicrospheres (1.3 g) having an enclosed drug ratio of 0.8%.

Preparation Example 3

Dichloromethane (20 mL) was added to poly(lactic-co-glycolic acid)(RESOMER RG504, produced by Evonik Industries, 2.0 g), which wasdissolved by using an ultrasonic washing machine, and the compound ofExample 23 (124 mg) was further added to the solution, and dissolved.This solution was added portionwise to a 0.1% aqueous solution ofpolyvinyl alcohol (300 mL) stirred at 3,000 rpm using a homomixer (MARKII produced by PRIMIX Corporation), and the mixture was stirred at roomtemperature for 10 minutes to obtain an o/w emulsion. This o/w emulsionwas stirred at room temperature for 16 hours, dichloromethane wasvolatilized to solidify the oil phase, and the resultant was centrifuged(3,000 rpm, 20° C., 15 minutes) by using a centrifugation machine. Afterthe supernatant was removed, the precipitates were dispersed in a 0.1%(w/v) solution of Tween 80, the dispersion was passed thorough sieves of53 μm and 20 μm, and the sample remained on the sieve of 20 μm wascentrifuged (3,000 rpm, 20° C., 15 minutes). The supernatant wasremoved, then purified water was added to the precipitates, the mixturewas centrifuged again (3,000 rpm, 20° C., 15 minutes), and thesupernatant was removed. The precipitates were frozen at −80° C., anddried under reduced pressure (48 hours) to obtain drug-containingmicrospheres (1.1 g) having an enclosed drug ratio of 3.7%.

Test Example 1: Measurement of EP₄ Agonist Activity

In order to investigate EP₄ receptor agonist activity of the compoundsof the present invention, production of cAMP was measured in HEK293cells that were made to stably express the human EP₄ receptor.

(1) Measurement Method

By using Refseq Database, prostaglandin E receptor was searched for. Asa result, gene information of human EP₄ (NM_000958) receptor wasobtained. On the basis of this sequence information, the human EP₄receptor gene was cloned by PCR in a conventional manner using humancDNA as the template, and HEK293 cells that were made to stably expressthe human EP₄ receptor were established. When cryopreserved cells ofthis strain were thawed and used, cells subcultured three times or morewithin a certain period of time (about 1 to 2 weeks) by using Dulbecco'sModified Eagle's Medium (Dulbecco's Modified Eagle's Medium may behenceforth abbreviated as DMEM) containing 10% FBS, 50 units each ofpenicillin and streptomycin were used. The subcultured cells wereinoculated in wells of a poly-D-lysine-coated 96-well plate at a densityof 2×10⁴ to 2.5×10⁴ cells/well, and cultured for one day. The medium inthe wells was removed by suction, then DMEM (80 μL) was added to eachwell, and incubation was performed at 37° C. for 15 minutes. Then, 20 μLof an assay medium (DMEM containing 100 mM HEPES and 1 mM IBMX)containing PGE₂ or a test compound (at a concentration 5 times higherthan the final concentration) was added to each well to start thereaction. The reaction was allowed at 37° C. for 30 minutes, then themedium was removed by suction, Assay/Lysis Buffer (100 μL) contained incAMP Screen Kit (produced by Applied Biosystems) was added to terminatethe reaction. Then, the reaction mixture was incubated at 37° C. for 30minutes to prepare a sample for quantification of cAMP, and the amountof cAMP in the sample was quantified according to the method indicatedin cAMP Screen Kit. By non-linear regression of the compoundconcentration and the amount of cAMP, the concentration of the compoundrequired for increasing cAMP to 50% of the maximum increase (EC₅₀ value)was calculated by using the Kaleida Graph.

(2) Measurement Results

As shown in Table 1, the compounds of the present invention showedsuperior EP₄ agonist activity. In particular, they showed superior EP₄agonist activity even compared with that of the compound of ComparativeExample 1, which is a known compound similar to the compounds of thepresent invention.

For the compounds for which the EP₄ agonist activity was measured two ormore times, average values are indicated, if needed. Exp. No. mentionedin the table means example number.

TABLE 1 Exp. No. EC₅₀ (nM) 1 0.045 2 0.035 3 0.13 4 0.34 5 0.3 6 0.56 70.09 8 0.08 9 0.09 10 0.065 11 0.09 12 0.6 13 0.34 14 0.3 15 0.63 16 0.417 0.026 18 0.1 19 0.1 20 0.07 21 0.5 22 0.055 23 0.033 24 0.3 25 0.09526 0.17 27 0.09 28 0.087 29 0.16 Comparative 2.5 Example 1

Test Example 2: Receptor Binding Test Using Human EP Receptor-ExpressingCells

In order to evaluate selectivity to each EP receptor subtype, [³H]PGE₂binding-inhibition activities of test compounds for cell membranes onwhich human EP₂, human EP₃, and human EP₄ receptors were stablyexpressed were measured.

(1) Measurement Method

As the membrane fractions of the prostaglandin E receptors EP₂, EP₃, andEP₄, 10.0 μg protein/tube each of HTS185M, HTS092M, and HTS142M of MerckMillipore were used, respectively. Each membrane fraction was incubatedwith a reaction mixture (250 μL/tube) containing a test compound and[³H]PGE₂ at 25° C. for 60 minutes. The final concentrations of [³H]PGE₂were 2.56 nmol/L in the EP₂ measurement system, 1.54 nmol/L in the EP₃measurement system, and 1.24 nmol/L in the EP₄ measurement system. Afterthe reaction, the membrane fraction was collected on filter paper byusing a cell harvester, the filter paper was transferred to ameasurement vial, and the measurement was performed on a liquidscintillation counter.

The nonspecific binding was determined as binding observed in thepresence of excess amount (10 μM) of unlabeled PGE₂. The measurement ofthe [³H]PGE₂ binding-inhibiting activity of a test compound wasperformed by adding the test compound at various concentrations. Thefollowing buffers were used for the reaction.

Buffer for EP₂: 50 mmol/L HEPES-NaOH (pH 7.4) containing 5 mmol/L MgCl₂,1 mmol/L CaCl₂, and 0.2% BSABuffer for EP₃: 50 mmol/L Tris-HCl buffer containing 10 mmol/L MgCl₂,and 1 mmol/L EDTABuffer for EP₄: 50 mmol/L HEPES-NaOH (pH7.4) containing 5 mmol/L MgCl₂,1 mmol/L CaCl₂, and 0.2% BSA

A dose-response curve for the [³H]PGE₂ binding-inhibiting activity wascreated for each test compound, and the concentration of the testcompound that inhibits 50% of the binding of PGE₂ and the receptor (IC₅₀value) was calculated.

(2) Measurement Results

As shown in Table 2, the compounds of the present invention showedsuperior EP₄ selectivity. Exp. No. mentioned in the table means examplenumber.

TABLE 2 IC₅₀ (nM) Exp. No. EP₂ EP₃ EP₄ 23 >1000 >1000 4.3524 >1000 >1000 43.0 25 >1000 >1000 10.9 26 >1000 >1000 16.2

Test Example 3: Neo-Osteogenesis Action in Rat Femur

In order to investigate osteogenesis-promoting action of the compoundsof the present invention, the compounds were allowed to act on ratfemurs, and the formed new bone was evaluated.

(1) Measurement Method

Eight weeks old female SD rats (Charles River Japan) were fixed in theside lying position under triple anesthesia (medetomidine hydrochloride,midazolam, and butorphanol tartrate). After hair of the left femoralregion was shaved with a hair clipper, and disinfection treatment wasperformed with 70% ethanol, an in situ-solidified gel solution of a testcompound, specifically, a test compound dissolved inpoly(lactic-co-glycolic acid) (RESOMER RG502H, produced by EvonikIndustries)/poly(lactic-co-glycolic acid)-polyethylene glycol blockcopolymer (5050 DLG mPEG 5000, produced by LakeshoreBiomaterials)/N-methyl-2-pyrroridone (produced by Wako) (weight ratio47%/3%/50%), was filled in a 1-mL syringe, a 21 G injection needle wasattached to the syringe, and the needle was transdermally stabbed fromthe quadriceps to the periosteum near the center of the femoraldiaphyseal. Then, 100 μg as the amount of the test compound, or 50 μL asthe administration volume of the solution was injected between thequadriceps and the periosteum, and the injection needle was drawn out.To the rats of the control group, the aforementioned in situ-solidifiedgel solution alone was administered. One week after the administrationof the drug solution, the triple anesthesia was given to the animals,and they were fixed in the supine position, and euthanized by bleeding.The left femur was extracted, circumferential tissues such as muscleswere removed, the bone mineral content of the whole femur was measuredby using a bone mineral analyzer DCS-600EX (produced by ALOKA), then itwas divided into three portions along the long axis, and the bonemineral content of the center part (diaphysis) was evaluated. The testwas performed with the groups each consisting of 6 animals.

(2) Measurement Results

In the groups administered with the typical compounds of the presentinvention, bone mineral content of the left femur diaphysis increasedcompared with the control group (Tables 3 to 5). On the other hand, thebone mineral content of the diaphysis of the right femur, to which thedrug solution was not administered, was not affected. On the basis ofthese results, it was confirmed that the compounds of the presentinvention are useful as a bone formation-promoting agent used by localadministration. For all the compound administration groups, death of theanimals was not observed, the side reactions observed for PGE₂administration was not observed, either, and thus it was demonstratedthat the compounds of the present invention can be safely administered.

The test results are mentioned for each test.

TABLE 3 Bone mineral content of femur ⅓ center region (mg) Left femurRight femur (no (administered administration Exp. No. with compound) ofcompound) Control 69.4 ± 3.3 62.9 ± 2.2 group 3 80.2 ± 2.6 66.7 ± 2.7 681.3 ± 3.6 64.7 ± 2.4 8 98.7 ± 4.1 68.0 ± 1.2 13 82.0 ± 3.4 65.5 ± 2.7

TABLE 4 Bone mineral content of femur ⅓ center region (mg) Left femurRight femur (no (administered administration Exp. No. with compound) ofcompound) Control 65.4 ± 1.9 61.6 ± 1.5 group 10 78.0 ± 1.5 61.1 ± 0.519 85.8 ± 2.3 59.6 ± 1.4 20 79.4 ± 5.2 58.7 ± 3.6 22 79.5 ± 1.9 60.3 ±2.7

TABLE 5 Bone mineral content of femur ⅓ center region (mg) Left femurRight femur (no (administered administration Exp. No. with compound) ofcompound) Control 68.0 ± 2.4 63.4 ± 2.1 group 23 82.7 ± 6.7 58.9 ± 2.924 80.7 ± 4.0 61.5 ± 2.3 25 79.1 ± 4.0 61.0 ± 2.1 26 87.0 ± 1.6 65.9 ±1.2

Test Example 4: Neo-Osteogenesis Action in Dog Femur

In order to investigate an osteogenesis-promoting action of thecompounds of the present invention, for influence of administration ofmicrospheres containing a test compound in the vicinity of the femur ofdog, osteogenesis-promoting action was evaluated by measuring new boneformed after the administration.

(1) Measurement Method

Nine to eleven months old female beagle dogs (KITAYAMA LABES) wereanesthetized by administration of a 1:1 mixture of ketaminehydrochloride (Ketalar 500 mg, Daiichi Sankyo Propharma Co., Ltd.) andxylazine (Selactar 2% Injection, Bayer Yakuhin, Ltd.) at a dose of about0.5 mL/kg, and isoflurane listed in Japanese Pharmacopoeia (Elucaine,Mylan Pharmaceutical) was used for maintenance anesthesia, which wasadministered with an inhaler IMPAC6 (VetEquip Inc.). After hair of thefemoral region of the right hind leg was shaved, and disinfectiontreatment was performed, 350 μL of a microsphere suspension obtained bysuspending microspheres (prepared according to the method of PreparationExample 1 or 2) containing a test compound (compound of Example 23) in aCMC solution was transdermally administered around the periosteum of thefemoral diaphysis by using a 1-mL injection syringe and a 21 G injectionneedle. The doses of the test compound were 0.01, 0.1, 1.0, 10, and 100μg/site, and the aforementioned microspheres were used in an amountcorresponding to each dose. As a control group, a drug liquid obtainedby suspending the microspheres not containing the test compound in 350μL of the CMC solution alone was administered. Four weeks after theadministration of the drug liquid, the animals were euthanized bybleeding under pentobarbital sodium (Somnopentyl) anesthesia. The femursof both sides were extracted, immersed in a 10% neutral bufferedformalin solution, and stored. The bone mineral content of the femur wasmeasured by using Discovery X-ray bone density analyzer (produced byToyo Medic). The test was performed with the groups each consisting of 4dogs

(2) Measurement Results

Four weeks after the administration of the aforementioned microspheresuspension to the femoral diaphyses of the dogs, the bone mineralcontent of the femurs increased with the doses of 1.0, 10, and 100 μg asthe administration amount of the test compound in a dose-dependentmanner compared with the control group (Table 6). On the basis of theseresults, it was confirmed that the compounds of the present inventionare useful as an osteogenesis-promoting agent used by localadministration. For all the compound administration groups, death of theanimals was not observed, the side reactions usually observed for PGE₂administration was not observed, either, and thus it was demonstratedthat the aforementioned microsphere preparation containing the compoundsof the present invention can be safely administered.

TABLE 6 Bone mineral content of femur ⅓ center region (g) Left femur (noRight femur administration of (administered compound) with compound)Control 2.6 ± 0.2 2.9 ± 0.3 group 0.01 μg 2.9 ± 0.4 2.9 ± 0.4 0.1 μg 2.6± 0.1 2.7 ± 0.2 1.0 μg 3.0 ± 0.1 4.1 ± 0.4 10 μg 2.8 ± 0.3 6.4 ± 0.7 100μg 2.7 ± 0.4 8.2 ± 1.1

Test Example 5: Fracture Healing-Promoting Action in Rat Femur ClosedFracture Model

In order to investigate fracture healing-promoting action of thecompounds of the present invention, influence of injection ofmicrospheres containing a test compound at a fracture site of a ratfemur closed fracture model was confirmed.

(1) Measurement Method

Thirteen weeks old female SD rats (Japan SLC) were fixed in the sidelying position under triple anesthesia (medetomidine hydrochloride,midazolam, and butorphanol tartrate). After hair of a region from theleft knee to the femoral region was shaved with a hair clipper, anddisinfection treatment was performed with povidone-iodine listed inJapanese Pharmacopoeia (Isocline solution for animals, 20 mg ofpovidone-iodine listed in Japanese Pharmacopoeia in 1 mL, Meiji SeikaPharma), the skin of the knee part and the medial great muscle of thelateral part of the patella were excised, and the patella was shiftedfrom the femoral head. A drill bit attached to a trephine was put on theintercondylar fossa of the exposed femoral head, and manually rotated tomake a bore. A Kirschner wire (Mizuho Co., Ltd.) having a diameter of1.2 mm preliminarily cut in a length of 31 mm was inserted into themedullary cavity of the femur through the bore. Then, the left femoralregion was fixed on a three-point bending test jig of a small desktopuniversal testing machine (EZ Test, Shimadzu Corp.), and closed fractureof the femoral diaphyseal was caused by giving a dynamic load. Whetherfracture was successfully introduced or not was confirmed by confirminggeneration of a complete transverse fracture in the femoral diaphysealon an X-ray image obtained with a soft X-ray generator (M-100W, Softex)and a digital X-ray sensor (NX-04, RF Co., Ltd.). A microspheresuspension obtained by suspending microspheres (prepared according tothe method of Preparation Example 3) containing a test compound(compound of Example 23) in a CMC solution was injected into thefracture part in a volume of 100 μL as the administration volume(containing 100 or 300 μg of the test compound). As the control group, adrug liquid obtained by suspending the microspheres not containing theaforementioned test compound in the CMC solution (100 μL) alone wasadministered. One, two, and three weeks after the generation offracture, soft X-ray images were taken under isoflurane anesthesia, andthe areas of the callus part in the X-ray images were quantified byusing Image J. Four weeks after the generation of fracture, soft X-rayimages were taken under triple anesthesia, the animals were fixed in thesupine position, and euthanized by bleeding, and the left femurs wereextracted. By confirming presence or absence of continuity of the callusunder blinding in the soft X-ray images obtained 4 weeks after thegeneration of fracture, bone union was determined. The femur sampleswere cryopreserved until implementation of a bone strength test, andtorsional strength was measured on the test day by using a bone strengthmeasurement apparatus (MZ-500S, Maruto Instrument Co., Ltd.).

This test can also be performed by using dogs instead of rats.

(2) Measurement Results

In the test compound administration group, two week after the generationof fracture and thereafter, increase of fracture callus area wasobserved (Table 7), and the bone union rate determined on the basis ofthe X-ray images obtained 25 days after the generation of fracture wasclearly improved (Table 8), compared with the control group. In thefemurs extracted from the animals of the test compound administrationgroup four weeks after the generation of fracture, increase of bonestrength determined by the torsion test (maximum torque) was observedcompared with the control group (Table 8). On the basis of theseresults, it was confirmed that the compounds of the present inventionare useful as a bone union-promoting agent in a fracture healingprocess. For all the compound administration groups, death of theanimals was not observed, the side reactions usually observed for PGE₂administration was not observed, either, and thus it was demonstratedthat the aforementioned microsphere preparation containing the compoundof the present invention can be safely administered.

TABLE 7 Callus area (mm²) One week after Two weeks after Three weeksafter fracture fracture fracture Control 1.98 ± 1.52 22.3 ± 4.1 27.4 ±4.4 group 100 μg 5.24 ± 2.87 28.6 ± 4.7 35.9 ± 6.3 300 μg 6.01 ± 2.7528.9 ± 3.8 33.8 ± 6.8

TABLE 8 Bone union rate after 25 days (%) Maximum torque (N · cm)Control group 61.1 18.8 ± 6.1 100 μg 90.0 20.4 ± 7.1 300 μg 85.7  26.3 ±11.7

Test Example 6: Bone Union-Promoting Action in Dog Lumbar VertebraePosterolateral Fixation Model

In order to investigate a bone union-promoting action of the compoundsof the present invention, influence of mixing of microspheres containinga test compound at the time of autologous bone grafting was confirmed byusing a lumbar vertebrae posterolateral fixation model.

(1) Measurement Method

Twelve to thirteen months old male beagle dogs (KITAYAMA LABES) wereanesthetized by administration of a 1:1 mixture of ketaminehydrochloride (Ketalar 500 mg, Daiichi Sankyo Propharma Co., Ltd.) andxylazine (Selactar 2% Injection, Bayer Yakuhin, Ltd.) at a dose of about0.5 mL/kg, and Japanese Pharmacopoeia-listed isoflurane (Elucaine,produced by Mylan Pharmaceutical) was inhaled with an inhaler IMPAC6(VetEquip Inc.) to maintain the anesthesia. The animal was fixed in theprone position, hair of a wide area over the right and left spina iliacaposterior superiors, part around iliac crest, and lower back was shaved,and disinfection treatment was performed with povidone-iodine listed inJapanese Pharmacopoeia (Isocline solution for animals, 20 mg ofpovidone-iodine listed in Japanese Pharmacopoeia in 1 mL, Meiji SeikaPharma) and ethanol for disinfection (Wako Pure Chemical Industries).The skin and soft tissue were cut and opened from the spina iliacaposterior superior along the iliac crest using a scalpel, and then themuscles covering the iliac crest were separated under the periosteum toexpose the iliac crest. About 2 g each of the right and left iliums wereextracted by using a rongeur and bone scissors, and astriction wasperformed. The collected ilium, from which the soft tissues wereremoved, was finely broken with bone scissors, and thereby made intochips of 1 mm size to obtain 2 g each of bone grafts for right and left.Then, the skin was dissected along the spinous processes of lower backwith a scalpel, the left and right lumbodorsal fascias were dissected,and then the transverse processes of the fourth and fifth lumbarvertebrae were exposed while the multifidus muscle and longissimusmuscle were separated and dissected between the fascias thereof. Thesoft tissues adhering to the transverse processes were removed, and thendecortication of the cortical bone on the surface of the transverseprocesses was performed with an electric drill (OS-40MV2, Osada ElectricCo., Ltd.) to prepare a bone graft bed. The bone graft (2 g) preparedabove was sufficiently mixed with a microsphere suspension obtained bysuspending microspheres containing a test compound (compound of Example23) in an amount corresponding to 10, 30, or 100 μg of the test compound(prepared according to the method of Preparation Example 1) in 800 μL ofa CMC solution, embedded and grafted on the transverse processes of thefourth and fifth lumbar vertebrae, and the bone graft bed between thetransverse processes. After the autologous bone grafting, thelumbodorsal fascia, subcutaneous tissues, and skin were sutured, and thesurgical site was disinfected. Twelve weeks after the operation, theanimals were euthanized with overdose (30 mg/kg) of sodiumpentobarbiturate (Somnopentyl, Kyoritsuseiyaku Corporation), and thenthe lumbar vertebrae were extracted. Bone union was judged by manuallyconfirming movability of the fourth and fifth lumbar vertebrae (manualpalpation) under blinding, and soft X-ray images were obtained from onedirection by using Softex M-60 (Softex). Each soft X-ray image wasevaluated by one person under blinding in accordance with the evaluationcriteria shown in Table 9. The test was performed with the groups eachconsisting of 5 animals.

TABLE 9 Grade Evaluation criteria 3 Osteogenesis advanced at thegrafting site, and the graft mass crosslinked the transverse processes,and fused to the transverse processes, vertebral arch, or centrum withuniform continuity. 2 Osteogenesis advanced at the grafting site, andthe graft mass crosslinked the transverse processes, and fused to thetransverse processes, vertebral arch, or centrum, but they did notconstitute one mass, and continuity between transverse processes wasonly partial, or gaps or radiolucent lines were observed in a part ofthe graft mass. 1 Advance of osteogenesis was observed mainly aroundtransverse processes, but the graft mass between the transverseprocesses did not have continuity, and definite gaps or radiolucentlines interrupting the graft mass were observed. 0 Osteogenesis was notobserved, and the graft material did not change, or was absorbed anddisappeared.

(2) Measurement Results

When lumbar vertebra samples were extracted, and osteogenesis wasevaluated on the basis of degree of calcification using soft X-rayimages, in the control group where the microspheres not containing thetest compound was mixed in the autologous graft ilium, movability wasobserved between the 4th and 5th lumbar vertebrae, but calcificationbetween transverse processes was not observed in the soft X-ray imagesin all the five examples. On the other hand, in the group where themicrospheres containing the test compound was administered, at each ofthe doses of 10, 30, or 100 μg, it was judged that movability wasobserved between the 4th and 5th lumbar vertebrae in one example out offive examples, but no movability was observed in the four otherexamples. In the soft X-ray images, promotion of osteogenes and osseouscontinuity were observed between the 4th and 5th lumbar vertebratransverse processes, namely, the continuity score was 2 or higher, atall the doses. In addition, the score increased in a dose-dependentmanner (Table 10, 2.4±0.5, 2.6±0.5, and 2.8±0.4, respectively).

On the basis of these results, it was confirmed that the compounds ofthe present invention are useful as an agent for promoting bone union inthe spine fixation based on autologous bone grafting. For all thecompound administration groups, death of the animals was not observed,the side reactions usually observed for PGE₂ administration was notobserved, either, and thus it was demonstrated that the aforementionedmicrospheres containing the compound of the present invention can besafely administered in the spine fixation.

TABLE 10 Control group 10 μg/site 30 μg/site 100 μg/site Manualevaluation of 0/5 4/5 4/5 4/5 bone union Continuity score 0 ± 0 2.4 ±0.5 2.6 ± 0.5 2.8 ± 0.4 based on soft X-ray image (Mean ± SD)

1.-12. (canceled)
 13. The compound mentioned below, or a salt thereof;


14. The compound mentioned below, or a salt thereof;


15. The compound mentioned below, or a salt thereof;


16. The compound mentioned below, or a salt thereof;


17. The compound mentioned below, or a salt thereof;


18. The compound mentioned below, or a salt thereof;


19. The compound mentioned below, or a salt thereof;


20. (canceled)
 21. A medicament containing the compound according to anyone of claims 13 to 19 or a pharmaceutically acceptable salt thereof asan active ingredient.
 22. The medicament according to claim 21, which isfor therapeutic treatment and/or promotion of healing of fracture. 23.The medicament according to claim 21, which is for promotion of boneunion in spinal fusion surgeries